Sulfonyl semicarbazides, semicarbazides and ureas,pharmaceutical compositions thereof, and methods for treating hemorrhagic fever viruses, including infections associated with arenaviruses

ABSTRACT

Compounds, methods and pharmaceutical compositions for treating viral infections, by administering certain novel semicarbazides, sulfonyl carbazides, ureas and related compounds in therapeutically effective amounts are disclosed. Methods for preparing the compounds and methods of using the compounds and pharmaceutical compositions thereof are also disclosed. In particular, the treatment and prophylaxis of viral infections such as caused by hemorrhagic fever viruses is disclosed, i.e., including but not limited to Arenaviridae (Junin, Machupo, Guanavito, Sabia and Lassa), Filoviridae (ebola and Marburg viruses), Flaviviridae (yellow fever, omsk hemorrhagic fever and Kyasanur Forest disease viruses), and Bunyaviridae (Rift Valley fever).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part and claims priority to U.S.patent application Ser. No. 11/791,926, which is a national stage entryunder U.S.C. 371(c), which claims priority to International PatentApplication Number PCT/US2005/043931, filed Dec. 6, 2005, which claimspriority to U.S. Provisional Patent Application Ser. No. 60/632,990,filed Dec. 6, 2004. All the applications are incorporated herein byreferencein the entirety and for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was supported in part by funds from the U.S. government(National Institutes of Health SBIR Grant Nos., R43 AI056525, and R44AI056525, Cooperative Research Agreement Grant No. U54 AI065357-06, andResearch Grant No. R01 AI074818-03) and the U.S. government maytherefore have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the use of sulfonyl semicarbazides,semicarbazides and ureas, as well as derivatives and analogs thereof,and pharmaceutical compositions containing the same, for the treatmentor prophylaxis of viral infections and diseases associated therewith. Inparticular, those viral infections and associated diseases caused byhemorrhagic fever viruses, such as Arenaviruses may be treated.

BACKGROUND OF THE INVENTION

Hemorrhagic fever viruses have been discussed in the scientificliterature. The following publications, patents and patent applicationsare cited in this application as superscript numbers:

The National Institute of Allergy and Infectious Diseases (NIAID) andthe Centers for Disease Control and Prevention (CDC) have classified anumber of viruses as potential agents of bioterrorism(www.bt.cdc.gov/agent/agentlist-category.asp). The highest threatagents, the Category A pathogens, have the greatest potential foradverse public health impact and mass casualties if used inill-intentioned ways. Within the Category A pathogens, there are anumber of viruses that can cause viral hemorrhagic fevers with high casefatality rates. The Category A hemorrhagic fever viruses pose seriousthreats as potential biological weapons because: 1) they can bedisseminated through aerosols; 2) a low dose (1-10 plaque forming unit(pfu)) can cause disease; 3) they cause severe morbidity and mortality(case fatality rates of 15-30%); 4) they can cause fear and panic in thegeneral public; 5) there are no U.S.-approved effective vaccines orspecific antivirals available; 6) these pathogens are easily availableand can be readily produced in large quantities; and 7) research onweaponizing various hemorrhagic fever viruses has been conducted.¹

Arenaviruses are enveloped viruses with a genome that consists of twosingle-stranded RNA segments designated small (S, 3.5 Kb) and large (L,7.5 Kb), both with an ambisense coding arrangement.³⁶ The S RNA segmentencodes the major structural proteins, nucleocapsid protein (NP) and aprecursor envelope protein (GPC) encoding two envelope glycoproteins(external GP1 and transmembrane GP2),^(18,24,30,31) and the L RNAsegment encodes the RNA polymerase protein L and an 11 KDa protein, Zprotein, with putative regulatory function.¹⁹ GP1 and GP2, which formthe tetrameric surface glycoprotein spike, are responsible for virusentry into targeted host cells.

The family Arenaviridae consists of a single genus (Arenavirus) thatincludes several viruses (currently 23 recognized viruses) causingsevere hemorrhagic fever diseases in humans.² The Arenaviridae familyhas been divided into two groups according to sequence-based phylogeny.The “Old World” group, originated from Africa, includes the humanpathogens lymphocytic choriomeningitis (LCM) virus and Lassa virus. The“New World” group, originated from Latin America, is divided into 3clades. Clade B includes in addition to Tacaribe and Amapari viruses,the Category A human pathogenic viruses Junin (Argentine hemorrhagicfever), Machupo (Bolivian hemorrhagic fever), Guanarito (Venezuelanhemorrhagic fever), and Sabiá (Brazilian hemorrhagic fever). TheseCategory A viruses are capable of causing severe and often fatalhemorrhagic fever disease in humans.

Rodents are the natural host of arenaviruses, although Tacaribe virus isfound in bats. The arenaviruses characteristically produce chronicviremic infections in their natural host,¹⁵ which in turn shed virus intheir urine and feces, ultimately infecting humans in close contact withthese infected materials either by aerosol or direct contact with skinabrasions or cuts. The natural history of the human disease isdetermined by the pathogenicity of the virus, its geographicaldistribution, the habitat and the habits of the rodent reservoir host,and the nature of the human-rodent interaction.²¹

Several Arenaviruses are associated with severe hemorrhagic disease inhuman. Lassa virus (from the Old World group) is responsible for Lassahemorrhagic fever, while 4 viruses from the New World group (all fromClade B) cause severe hemorrhagic fever in human. Those viruses are:Junin virus responsible for Argentine hemorrhagic fever, Machupo virusfor Bolivian hemorrhagic fever and Guanarito virus for Venezuelanhemorrhagic fever. Sabia virus was isolated from a fatal case ofhemorrhagic fever in Brazil. It is estimated that Lassa virus causes100,000-300,000 infections and approximately 5,000 deaths annually.⁵ Sofar an estimated 30,000 confirmed cases of Junin infections have beendocumented, while about 2,000 of Machupo, 200 of Guanarito and only 2 ofSabia.¹

Recent concerns over the use of Arenaviruses as biological weapons haveunderscored the necessity of developing small molecule therapeutics thattarget these viruses.¹ The availability of antiviral drugs directed atthese viruses would provide treatment and a strong deterrent againsttheir use as biowarfare agents. Since antiviral drugs can be easilyadministered (oral pill or liquid) and exert their antiviral effectwithin hours of administration, they will serve to effectively treatdiseased patients, protect those suspected of being exposed to thepathogen (post-exposure prophylaxis), and assist in the timelycontainment of an outbreak.

Currently, there are no virus-specific treatments approved for useagainst Arenavirus hemorrhagic fevers. Present disease managementconsists of general supportive care: monitoring and correcting fluid,electrolyte and osmotic imbalances and treating hemorrhaging withclotting factor or platelet replacement. Convalescent immune serumtherapy may be effective in treating cases of Junin and Machupo virusdisease, but the availability of such serum is extremely limited.

Ribavirin, a nucleoside analog, has been used with some success in Lassafever patients. In small trials, intravenous ribavirin given to patientswithin the first 6 days after development of fever decreased mortalityfrom 76% to 9%.⁷⁻⁹ A controlled trial of 18 patients with Argentinehemorrhagic fever resulted in 13% mortality in treated patients comparedwith 40% in untreated patients.¹⁰ Ribavirin therapy is associated withadverse effects including a dose-related, reversible hemolytic anemia and also has demonstrated teratogenicity and embryo lethality in severalanimal species. It is therefore classified as a pregnancy category Xdrug, contraindicated during pregnancy. Intravenous ribavirin isavailable in limited supplies in the U.S. for compassionate use under anFND application. The dosing regimen for ribavirin therapy that has beenused in cases of Lassa fever consists of an initial 30 mg/kg intravenous(IV) loading dose, followed by 16 mg/kg IV every 6 hours for 4 days;then 8 mg/kg IV every 8 hours for 6 days (total treatment time 10 days).The cost of treatment for an adult male is approximately $800. Theattributes f ribavirin make it less than ideal for the treatment ofArenavirus hemorrhagic fevers.

A number of in vitro inhibitors of Arenavirus replication have beenreported in the literature including phenothiazines, trifluoroperazineand chlorpromazine,¹ amantadine,^(12,13) brassinosteroids¹⁴ andactinomycin D.¹⁵ The anti-Arenavirus activities of these compounds aregenerally weak and non-specific.

The only Arenavirus hemorrhagic fever for which studies have beenundertaken toward development of a vaccine has been Argentinehemorrhagic fever (AHF) caused by Junin virus. A live-attenuatedvaccine, called Candid 1, has been evaluated in controlled trials amongagricultural workers in AHF-endemic areas, where it appeared to reducethe number of reported AHF cases with no serious side effects.¹⁶ It isnot known if the Candid 1 vaccine would be useful against otherArenavirus hemorrhagic fevers and this vaccine is not available in theUnited States of America.

Tacaribe virus is a biosafety level 2 (BSL 2) New World arenavirus (NWA)that is found in Glade B and phylogenetically related to the Category ANWA (Junin, Machupo, Guanarito and Sabiá). Tacaribe virus is 67% to 78%identical to Junin virus at the amino acid level for all four viralproteins. In order to screen for inhibitors of NWA a high-throughputscreening (HTS) assay for virus replication was developed using Tacaribevirus as a surrogate for Category A NWA. A 400,000 small moleculelibrary was screened using this HTS assay. A lead series was chosenbased on drug properties and this series was optimized through iterativechemistry resulting in the identity of a highly active and specificsmall molecule inhibitor of Tacaribe virus with selective activityagainst human pathogenic NWA (Junin, Machupo, Guanarito and Sabiá). Thismolecule demonstrates favorable pharmacodynamic properties whichpermitted the demonstration of in vivo anti-arenavirus activity in anewborn mouse model.

All human pathogens Arenaviruses from the New World group causinghemorrhagic fever are from the Clade B. These human pathogen virusesrequire manipulation under high-level containment (BSL-4). However,Amapari and Tacaribe viruses also from Clade B can be grown in tissueculture under BSL-2 (low-level) containment. Working under low-levelcontainment makes experimentations easier and safer with these viruses.While Amapari virus produces low cytopathic effect, Tacaribe virus canbe grown readily in cell culture and produce robust CPE in 4 to 6 days.Since this CPE is directly related to viral replication, compounds thatinhibit virus replication in cell culture can be identified readily asconferring protection from virus-induced CPE (although it istheoretically possible to inhibit CPE without inhibiting virusreplication). Moreover, compounds having identified activity againstTacaribe virus will also likely be active against Arenavirus humanpathogen causing hemorrhagic fever (Junin, Machupo, Guanarito and Sabia)given the high degree of homology (around 70% identity for all 4proteins of Tacaribe virus compared to Junin virus, with long stretch ofprotein with perfect identity) between these viruses.

What is needed in the art are new therapies and preventives for thetreatment of viral infections and associated diseases, such as caused byhemorrhagic fever viruses like Arenaviruses.

SUMMARY OF THE INVENTION

The present invention provides compounds and compositions and/or methodsfor the treatment and prophylaxis of viral infections, as well asdiseases associated with viral infections in living hosts. Inparticular, the present invention provides compounds and compositionsand/or methods for the treatment and prophylaxis of hemorrhagic feverviruses, such as Arenaviruses.

In one embodiment, the invention relates to a method for the treatmentor prophylaxis of a viral infection or disease associated therewith,comprising administering in a therapeutically effective amount to amammal in need thereof, a compound of formula I or a pharmaceuticallyacceptable salt thereof. In another embodiment, the invention relates toa pharmaceutical composition that comprises a pharmaceutically effectiveamount of the compound or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier. In addition, the inventionalso relates to compounds of formula I, as well as pharmaceuticallyacceptable salts thereof.

Preferred compounds of formula I include:

whereinn is an integer from 0-6;m is an integer from 0-1;p is an integer from 0-1;R₁ is selected from the group consisting of H and alkyl; R2 is selectedfrom the group consisting of substituted or unsubstituted phenyl,substituted and unsubstituted aryl, substituted and unsubstitutedheteroaryl, substituted or unsubstituted alkyl, substituted orunsubstituted branched alkyl, and substituted or unsubstitutedunsaturated cycloheteroalkyls;or where R1 and R2 combine together to form a substituted orunsubstituted C4-10 cyclic saturated heteroalkyl;R₃ is selected from the group consisting of H and alkyl;or a pharmaceutically acceptable salt thereof.Other compounds of formula I include:

whereinR₂ is selected from the group consisting of substituted or unsubstitutedphenyl, substituted and unsubstituted aryl, substituted andunsubstituted heteroaryl, substituted or unsubstituted alkyl,substituted or unsubstituted branched alkyl, and substituted orunsubstituted unsaturated cycloheteroalkylsor a pharmaceutically acceptable salt thereofFurther compounds of formula I include:

whereinR₁ is selected from the group consisting of H and alkyl;R₂ is selected from the group consisting of substituted or unsubstitutedphenyl, substituted and unsubstituted aryl, substituted andunsubstituted heteroaryl, substituted or unsubstituted alkyl,substituted or unsubstituted branched alkyl, and substituted orunsubstituted unsaturated cycloheteroalkyls;or where R₁ and R₂ combine together to form a substituted orunsubstituted C₄₋₁₀ cyclic saturated heteroalkyl;or a pharmaceutically acceptable salt thereof

In other embodiments, in the compound of formula I, n is 0 or 1. Also,in other embodiments, in the compound of formula I, m is 1 and p is 1 oralternatively, m is 0 and p is 0.

In further embodiments, in Formula I, R₁ and R₂ combine together to forma substituted or unsubstituted C₄₋₁₀ cyclic saturated heteroalkylselected from the group consisting of:

In still further embodiments, in Formula I, R₂ is selected from thegroup consisting of:

wherein each of R₅, R₆, R₇, R₈ and R₉ is independently selected from thegroup consisting of: hydrogen, acetyl, methoxy, trifluoromethyl, fluoro,chloro, bromo, iodo, acylamino, methyl, sulfonamide, trifluoromethoxy,carboxy, cyano and 1,1,2,2-tetrafluoroethoxy.

In particular, certain embodiments relate to a compound of formula Iselected from the group consisting of:

-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-(phenyl)-phenylsulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-(2-methyl-2-propyl)-phenylsulfonyljhydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[7-(4-methyl-3,4-dihydro-2H-benzo[1,4]oxazinyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[5-(1-dimethylamino-naphthyl)    sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2,4,6-trimethylphenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3-chloro-6-methoxyphenyl)    sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3,6-dimethoxyphenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-(4-[1,2,3]thiadiazolyl)phenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3-bromophenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-bromophenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-methylphenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-methoxyphenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-difluoromethoxyphenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3-fluoro-4-chloro-phenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,33-Hexafluoro-2-methylpropyl)-2-[(4-trifluoromethoxyphenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-fluoro-phenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3-methoxyphenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2-methylphenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3-trifluoromethylphenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2,4-dimethoxyphenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[2-(5-chloro-1,3-dimethyl-1H-pyrazolyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3-methylphenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-trifluoromethylphenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2-trifluoromethylphenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[4-(pyrrolidin-1-sulfonyl)phenyl    sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2-chlorophenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[2-(5-morpholin-4-yl)pyridyl    sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-(2-trifluoromethoxyphenyl)    sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2,4-dichlorophenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[phenylsulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3-difluoromethoxyphenyl)    sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3-cyanophenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-cyanophenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[5-(2,3-dihydrobenzo[1,4]dioxinyl)    sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-methylphenyl)sulfonyl]-1-methylhydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3-fluorophenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3,4-difluorophenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2,4-dimethylthiazol-5-yl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-acetylphenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2,6-difluorophenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2-fluorophenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2,5-difluorophenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-methylphenyl)sulfonyl]-2-methylhydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2,6-dichlorophenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2,6-ditrifluoromethylphenyl)    sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-methylphenyl)sulfonyl]hydrazine-1-methylcarboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-memylpropyl)-2-[(3,5-dimethylisoxazol-5-yl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-nitrophenyl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(1-methylimidazol-4-yl)sulfonyl]hydrazine-1-carboxamide;-   N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[methylsulfonyl]hydrazine-1-carboxamide;-   4-Phenylpiperazine-1-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-carboxamide;-   4-Morpholino-1-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-carboxamide;-   1-(2-Acetylphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-Piperidino-1-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-carboxamide;-   1-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-3-(3,4,5-trimethoxyphenyl)-urea;-   1-(4-Trifluoromethylphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   4-Methylpiperazine-1-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-carboxamide;-   1-Naphthalen-1-yl-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(4-Chlorophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   4-Phenylpiperidin-1-yl-1-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-carboxamide;-   1-(2-Phenyl(phenyl))-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(2,6-Difluorophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   2-[3-(1,1-Bis-trifluoromethylethyl)-ureido]benzamide;-   1-(2-Chloro-6-fluorophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(3-Trifluoromethylphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   2-[3-(1,1-Bis-trifluoromethylethyl)-ureido]benzenesulfonamide;-   1-(2,2,3,3-Tetrafluoro-2,3-dihydrobenzo[1,4]dioxin-5-yl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(3-Trifluoromethoxyphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(4-Trifluoromethoxyphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   4-Methyl-1-piperidine-1-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-carboxamide;-   1-Naphthalen-2-yl-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(2-fluorophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(2,6-Dimethoxyphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   3-Trifluormethoxy-4-[3-(1,1-bis-trifluoromethylethyl)-ureido]benzoicacid;-   1-Phenyl-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(3-Cyanophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(3-Methoxyphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(2-(1,1,2,2-Tetrafluoroethoxy)phenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   3-[3-(1,1-Bis-trifluoromethylethyl)-ureido]benzenesulfonamide;-   1-(3-fluorophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(4-Bromophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(2-Cyanophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(4-Cyanophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(2,2-Difluorobenzo[1,3]dioxol-4-yl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(4-Chlorophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(3-Methylphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   4-[3-(1,1-Bis-trifluoromethylethyl)-ureido]benzenesulfonamide;-   1-(2,6-Dibromophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(2-Methylphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(4-Methylphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-Pyrrolidinyl-1-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-carboxamide;-   1-(4-Fluorophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(2,4-Dibromophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   Azepane-1-carboxylic acid    (2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-amide;-   1-(4-Bromo-2-trifluoromethoxyphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(2-Trifluoromethoxyphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(2-Trifluoromethylphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;-   1-(2-Methoxyphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea;    and-   N-2-(1,1,1,3,3,3-hexafluoro-1-methylpropyl)-2-[(4-difluoromethoxyphenyl)sulfonyl]hydrazine-1-carboxamide.

The instant invention further provides for methods of treatingArenavirus infection, comprising administering in a therapeuticallyeffective amount to a mammal in need thereof, a compound of formula Ibelow:

-   -   wherein    -   n is an integer from 0-6;    -   m is an integer from 0-1;    -   p is an integer from 0-1;    -   R₁ is selected from the group consisting of H and alkyl;    -   R₂ is selected from the group consisting of substituted or        unsubstituted phenyl, substituted and unsubstituted aryl,        substituted and unsubstituted heteroaryl, substituted or        unsubstituted alkyl, substituted or unsubstituted branched        alkyl, and substituted or unsubstituted unsaturated        cycloheteroalkyls;    -   or where R₁ and R₂ combine together to form a substituted or        unsubstituted C₄₋₁₀ cyclic saturated heteroalkyl;    -   R₃ is selected from the group consisting of H and alkyl;    -   R₄ is selected from the group consisting of H and alkyl; and    -   wherein compound of formula I is selected from the group        consisting of:        1-[1,1-bis(trifluoromethyl)propyl]-3-[(2,4,6-trimethylphenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[4-tert-butylphenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(2,5-dimethoxyphenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-(2-naphthylsulfonylamino)urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-(4-isopropylphenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(5-chloro-2-methoxy-phenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(2-phenylphenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(3,4-dichlorophenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-bromophenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[[4-(difluoromethoxy)phenyl]sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(3,5-dimethylphenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[[5-(dimethylamino)-1-naphthyl]sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-(1-naphthylsulfonylamino)urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-methyl-2,3-dihydro-1,4-benzoxazin-7-yl)sulfonylamino]urea;    -   1-[[2,5-bis(2,2,2-trifluoroethoxy)phenyl]sulfonylamino]-3-[1,1-bis(trifluoromethyl)propyl]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[4-(thiadiazol-4-yl)phenyl]sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(5-bromo-2-methoxy-phenyl)sulfonylamino]urea;    -   and        1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-phenylphenyl)sulfonylamino]urea.

In yet another embodiment, the method of treatment according to theinstant invention comprises compound1-[1,1-bis(trifluoromethyl)propyl]-3-[[4-(thiadiazol-4-yl)phenyl]sulfonylamino]urea.

The present invention provides for a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a pharmaceuticallyeffective amount of a compound of formula I:

wherein

-   -   n is an integer from 0-6;    -   m is an integer from 0-1;    -   p is an integer from 0-1;    -   R₁ is selected from the group consisting of H and alkyl;    -   R₂ is selected from the group consisting of substituted or        unsubstituted phenyl, substituted and unsubstituted aryl,        substituted and unsubstituted heteroaryl, substituted or        unsubstituted alkyl, substituted or unsubstituted branched        alkyl, and substituted or unsubstituted unsaturated        cycloheteroalkyls;    -   or where R₁ and R₂ combine together to form a substituted or        unsubstituted C₄₋₁₀) cyclic saturated heteroalkyl;    -   R₃ is selected from the group consisting of H and alkyl;    -   R₄ is selected from the group consisting of H and alkyl; and        wherein the compound of formula I is selected from the group        consisting of:    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(2,4,6-trimethylphenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-tert-butylphenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(2,5-dimethoxyphenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-(2-naphthylsulfonylamino)urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-(4-isopropylphenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(5-chloro-2-methoxy-phenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(2-phenylphenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(3,4-dichlorophenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-bromophenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[[4-(difluoromethoxy)phenyl]sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(3,5-dimethylphenyl)sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[[5-(dimethylamino)-1-naphthyl]sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-(1-naphthylsulfonylamino)urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-methyl-2,3-dihydro-1,4-benzoxazin-7-yl)sulfonylamino]urea;    -   1-[[2,5-bis(2,2,2-trifluoroethoxy)phenyl]sulfonylamino]-3-[1,1-bis(trifluoromethyl)propyl]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[[4-(thiadiazol-4-yl)phenyl]sulfonylamino]urea;    -   1-[1,1-bis(trifluoromethyl)propyl]-3-[(5-bromo-2-methoxy-phenyl)sulfonylamino]urea;    -   and        1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-phenylphenyl)sulfonylamino]urea.

The present invention also provides for a pharmaceutical compositioncomprising compound1-[1,1-bis(trifluoromethyl)propyl]-3-[[4-(thiadiazol-4-yl)phenyl]sulfonylamino]urea.

The present invention also provides for the following compounds:1-[1,1-bis(trifluoromethyl)propyl]-3-[(2,4,6-trimethylphenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-tert-butylphenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(2,5-dimethoxyphenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-(2-naphthylsulfonylamino)urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-isopropylphenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(5-chloro-2-methoxy-phenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(2-phenylphenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(3,4-dichlorophenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-bromophenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-34[4-(difluoromethoxy)phenyl]sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(3,5-dimethylphenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[[5-(dimethylamino)-1-naphthyl]sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-(1-naphthylsulfonylamino)urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-methyl-2,3-dihydro-1,4-benzoxazin-7-yl)sulfonylamino]urea;1-[[2,5-bis(2,2,2-trifluoroethoxy)phenyl]sulfonylamino]-3-[1,1-bis(trifluoromethyl)propyl]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[[4-(thiadiazol-4-yl)phenyl]sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(5-bromo-2-methoxy-phenyl)sulfonylamino]urea;and1-[1,1-bis(trifluoromethyl)propyl]-3-(4-phenylphenyl)sulfonylamino]urea.

The present invention also provides for methods to detect and quantitatean arenavirus in a biological sample comprising incubating ST-375 with abiological sample; and further detecting and quantitating specificbinding of ST-375 to an arenavirus envelope glycoprotein, and thusdetecting and quantitating said arenavirus. In another embodiment, thebiological sample is selected from the group consisting of blood, serumand plasma. In yet another embodiment, the ST-375 binding to thearenavirus envelope glycoprotein is detected by fluorescence.

The present invention also provides for kits for detection andquantitation of an arenavirus in a biological sample comprising acontainer having ST-375; and further comprising instructions for usingsaid ST-375 to detect and quantitate the presence of arenavirus in thesaid biological sample. In one embodiment, said biological sample isselected from the group consisting of blood, serum and plasma.

In one embodiment, the mammal being treated is a human. In particularembodiments, the viral infection being treated is a hemorrhagic fevervirus, such as an Areanvirus. The Arenavirus may be selected from thegroup consisting of Junin, Machupo, Guanavito, Sabia, and Lassa.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the methods and formulations as more fully described below.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 provides the chemical structure, formula, and molecular weight ofST-336.

FIG. 2A and 2B show the effect of the time of addition of ST-336 onTacaribe virus yield and plaque formation. In FIG. 2A, Vero cells wereinfected with Tacaribe virus at a MOI=0.01. ST-336 was added prior to orduring Tacaribe infection (−1, 3, 6, 9, 12, 15, 18 or 21 hrs p.i.). At24 hrs p.i., virus yields were determined by plaque assay. In FIG. 2B,Vero cells were infected with 400 pfu Tacaribe virus. ST-336 was addedfor 1 hour before the infection (−1), for 1 hour during adsorption (0),and for 1 hour after the infection (+1). Infected monolayers were washedwith PBS and overlayed with medium containing agarose. Five dayspost-infection, cells were glutaraldehyde fixed and crystal violetstained prior to plaque counting.

FIG. 3 shows that ST-336 binds with slow Koff to intact Tacaribe virionin the absence of cells. In FIG. 3A, a diagram of the virus dilutionscheme prior to plating is provided. The virus mixed with ST-336 anddiluted (left side) or virus diluted and ST-336 added after dilution(right side). In FIG. 3B, pictures of the plaques that resulted afterplating each dilution shown in FIG. 3A on Vero cells is provided.

FIG. 4 shows the mapping of ST-336 drug resistant variants (“DRVs”). InFIG. 4A, a linear map of the glycoprotein precursor (“GPC”) showing thelocation of the signal peptide (“SP”), transmembrane domain (“TM”), thecleavage site between GP1 and GP2 (K261-A262), the location of the fourST-336 resistant mutants (“DR #1-4”), and the amino acid change for eachis provided. In FIG. 4B, the amino acid sequence alignment of GP2 fromwild type NWA and ST 336 DRVs is shown. Shown is the amino acid sequenceof the C-terminal portion of GP2 (amino acids 397 to 457) containing thetransmembrane domain (marked by vertical lines), the location of themutations for DR#1-4 (underlined), and the amino acid difference inAmapari (in bold).

FIG. 5 provides the chemical structure, formula, and molecular weightfor ST-294.

FIG. 6 shows the effect of ST-294 in newborn mice challenged withTacaribe virus. Four day old BALB/c mice were infected IP with 30×LD50Tacarbide virus and treated daily for 10 days with vehicle (control),ribavarin at 25 mg/kg, ST-294 twice a day (BID) at 50 mg/kg or once aday (SID) at 100 mg/kg. Shown in FIG. 6 are the percent survivors ineach treatment group on day 9 and day 10 after infection.

FIG. 7 shows chemical structures of fusion inhibitors.

FIG. 8 shows immunoprecipitation of insect and mammalian-cell derivedcleavage-defective GPC.

FIG. 9 shows biosensor analysis (Biacore T100) of the interaction ofsmall-molecule fusion inhibitors with detergent solubilized icd-GPC.

FIG. 10 shows inhibition of pH-induced cell-cell fusion.

FIG. 11 shows competitive binding of small-molecule fusion inhibitors toicd-GPC.

DETAILED DESCRIPTION OF THE INVENTION

As above, this invention relates to compounds which are useful for thetreatment and prophylaxis of viral infections, as well as diseasesassociated with viral infections in living hosts. In particular, thepresent invention provides compounds and compositions and/or methods forthe treatment and prophylaxis of hemorrhagic fever viruses, such asArenaviruses. However, prior to describing this invention in furtherdetail, the following terms will first be defined.

DEFINITIONS

In accordance with this detailed description, the followingabbreviations and definitions apply. It must be noted that as usedherein, the singular forms “a”, “an”, and “the” include plural referentsunless the context clearly dictates otherwise.

The publications discussed herein are provided solely for theirdisclosure. Nothing herein is to be construed as an admission that thepresent invention is not entitled to antedate such publication by virtueof prior invention. Further, the dates of publication provided may bedifferent from the actual publication dates, which may need to beindependently confirmed.

Where a range of values is provided, it is understood that eachintervening value is encompassed within the invention. The upper andlower limits of these smaller ranges may independently be included inthe smaller, subject to any specifically excluded limit in the statedrange. Where the stated range includes one or both of the limits, rangesexcluding either both of those included limits are also included in theinvention. Also contemplated are any values that fall within the citedranges.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

By “patient” or “subject” is meant to include any mammal A “mammal”, forpurposes of treatment, refers to any animal classified as a mammal,including but not limited to humans, domestic and farm animals, and zoo,sports, or pet animals, such as dogs, horses, cats, cows, and the like.Preferably, the mammal is human.

The term “efficacy” as used herein in the context of a chronic dosageregime refers to the effectiveness of a particular treatment regime.Efficacy can be measured based on change the course of the disease inresponse to an agent of the present invention.

The term “success” as used herein in the context of a chronic treatmentregime refers to the effectiveness of a particular treatment regime.This includes a balance of efficacy, toxicity (e.g., side effects andpatient tolerance of a formulation or dosage unit), patient compliance,and the like. For a chronic administration regime to be considered“successful” it must balance different aspects of patient care andefficacy to produce the most favorable patient outcome.

The terms “treating”, “treatment”, and the like are used herein to referto obtaining a desired pharmacological and physiological effect. Theeffect may be prophylactic in terms of preventing or partiallypreventing a disease, symptom or condition thereof and/or may betherapeutic in terms of a partial or complete cure of a disease,condition, symptom or adverse effect attributed to the disease. The term“treatment”, as used herein, covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseasefrom occurring in a subject which may be predisposed to the disease buthas not yet been diagnosed as having it, i.e., causing the clinicalsymptoms of the disease not to develop in a subject that may bepredisposed to the disease but does not yet experience or displaysymptoms of the disease; (b) inhibiting the disease, i.e., arresting orreducing the development of the disease or its clinical symptoms; or (c)relieving the disease, i.e., causing regression of the disease and/orits symptoms or conditions. The invention is directed towards treating apatient's suffering from disease related to pathological inflammation.The present invention is involved in preventing, inhibiting, orrelieving adverse effects attributed to pathological inflammation overlong periods of time and/or are such caused by the physiologicalresponses to inappropriate inflammation present in a biological systemover long periods of time.

As used herein, “acyl” refers to the groups H—C(O)—, alkyl-C(O)—,substituted alkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—,alkynyl-C(O)-, substituted alkynyl-C(O)— cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—,substituted heteroaryl-C(O), heterocyclic-C(O)—, and substitutedheterocyclic-C(O)— wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Acylamino” refers to the group —C(O)NRR where each R is independentlyselected from the group consisting of hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, substituted heterocyclic and whereeach R is joined to form together with the nitrogen atom a heterocyclicor substituted heterocyclic ring wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Alkenyl” refers to alkenyl group preferably having from 2 to 10 carbonatoms and more preferably 2 to 6 carbon atoms and having at least 1 andpreferably from 1-2 sites of alkenyl unsaturation.

“Lower alkenyl” refers to an alkenyl group preferably having from 2 to 6carbon atoms and having at least 1 site and preferably only 1 site ofalkenyl unsaturation (i.e., >C=C<). This term is exemplified by groupssuch as allyl, ethenyl, propenyl, butenyl, and the like.

“Substituted alkenyl” refers to alkenyl groups having from 1 to 5substituents independently selected from the group consisting of alkoxy,substituted alkoxy, acyl, acylamino, thiocarbonylamino, acyloxy, amino,amidino, alkylamidino, thioamidino, aminoacyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aryl, substituted aryl,aryloxy, substituted aryloxy, aryloxyaryl, substituted aryloxyaryl,halogen, hydroxyl, cyano, nitro, carboxyl, carboxylalkyl,carboxyl-substituted alkyl, carboxyl-cycloalkyl, carboxyl-substitutedcycloalkyl, carboxylaryl, carboxyl-substituted aryl, carboxylheteroaryl,carboxyl-substituted heteroaryl, carboxylheterocyclic,carboxyl-substituted heterocyclic, cycloalkyl, substituted cycloalkyl,guanidino, guanidinosulfone, thiol, thioalkyl, substituted thioalkyl,thioaryl, substituted thioaryl, thiocycloalkyl, substitutedthiocycloalkyl, thioheteroaryl, substituted thioheteroaryl,thioheterocyclic, substituted thioheterocyclic, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy,substituted cycloalkoxy, heteroaryloxy, substituted heteroaryloxy,heterocyclyloxy, substituted heterocyclyloxy, oxycarbonylamino,oxythiocarbonylamino, cycloalkyloxy, substituted cycloalkyloxy,heteroaryloxy, substituted heteroaryloxy, —OS(O)₂-alkyl,—OS(O)₂-substituted alkyl, OS(O)₂-aryl, —OS(O)₂-substituted aryl,—OS(O)₂-heteroaryl, —OS(O)₂-substituted heteroaryl,—OS(O)₂-heterocyclic, —OS(O)₂-substituted heterocyclic, —OSO₂—NRR whereR is hydrogen or alkyl, —NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl,—NRS(O)₂-aryl, —NRS(O)₂—substituted aryl, —NRS(O)₂-heteroaryl,—NRS(O)₂-substituted heteroaryl, —NRS(O)₂—heterocyclic,—NRS(O)₂-substituted heterocyclic, —NRS(O)₂—NR-alkyl, —NRS(O)₂—NR—substituted alkyl, —NRS(O)₂—NR-aryl, —NRS(O)₂—NR-substituted aryl,—NRS(O)₂—NR-heteroaryl, —NRS(O)₂—NR-substituted heteroaryl,—NRS(O)₂—NR-heterocyclic, —NRS(O)₂—NR-substituted heterocyclic where Ris hydrogen or alkyl, mono- and di-alkylamino, mono- and di-(substitutedalkyl)amino, mono- and di-arylamino, mono- and di-substituted arylamino,mono- and di-heteroarylamino, mono- and di-substituted heteroarylamino,mono- and di-heterocyclic amino, mono- and di-substituted heterocyclicamino, unsymmetric di-substituted amines having different substituentsindependently selected from the group consisting of alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic, substituted heterocyclic and substituted alkenyl groupshaving amino groups blocked by conventional blocking groups such as Boc,Cbz, formyl, and the like or alkenyl/substituted alkenyl groupssubstituted with —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-alkenyl,—SO₂-substituted alkenyl, —SO₂-cycloalkyl, —SO₂— substituted cycloalkyl,—SO₂-aryl, —SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substitutedheteroaryl, —SO₂-heterocyclic, —SO₂-substituted heterocyclic and —SO₂NRRwhere R is hydrogen or alkyl.

Preferably, the substituents are independently selected from the groupconsisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminocarbonylamino,aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryloxy,carboxyl, carboxyl esters, cyano, cycloalkyl, substituted cycloalkyl,cycloalkyloxy, substituted cycloalkyloxy, halogen, heteroaryl,substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy,heterocyclic, substituted heterocyclic, hydroxyl, nitro, andoxycarbonylamino.

“Alkoxy” refers to the group “alkyl-O-” which includes, by way ofexample, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, tert-butoxy,sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

“Substituted alkoxy” refers to the group “substituted alkyl-O-”.

“Alkyl” refers to linear or branched alkyl groups preferably having from1 to 10 carbon atoms and more preferably 1 to 6 carbon atoms. This termis exemplified by groups such as methyl, t-butyl, n-heptyl, octyl andthe like.

“Lower alkyl” refers to monovalent alkyl groups having from 1 to 5carbon atoms including straight and branched chain alkyl groups. Thisterm is exemplified by groups such as methyl, ethyl, iso-propyl,n-propyl, n-butyl, sec-butyl, t-butyl, n-pentyl and the like. “Loweralkyl” may be optionally substituted with a halogen, such as chloro,fluoro, bromo and the like.

“Substituted alkyl” refers to an alkyl group, of from 1 to 10 carbonatoms, having from 1 to 5 substituents independently selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,thiocarbonylamino, acyloxy, amino, amidino, alkyl amidino, thioamidino,aminoacyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aryl, substituted aryl, aryloxy, substituted aryloxy, aryloxylaryl,substituted aryloxyaryl, cyano, halogen, hydroxyl, nitro, carboxyl,carboxylalkyl, carboxyl-substituted alkyl, carboxyl-cycloalkyl,carboxyl-substituted cycloalkyl, carboxylaryl, carboxyl-substitutedaryl, carboxylheteroaryl, carboxyl-substituted heteroaryl,carboxylheterocyclic, carboxyl-substituted heterocyclic, cycloalkyl,substituted cycloalkyl, guanidino, guanidinosulfone, thiol, thioalkyl,substituted thioalkyl, thioaryl, substituted thioaryl, thiocycloalkyl,substituted thiocycloalkyl, thioheteroaryl, substituted thioheteroaryl,thioheterocyclic, substituted thioheterocyclic, heteroaryl, substitutedaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic,cycloalkoxy, substituted cycloalkoxy, heteroaryloxy, substitutedheteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy,oxycarbonylamino, oxythiocarbonylamino, cycloalkyloxy, substitutedcycloalkyloxy, heteroaryloxy, substituted heteroaryloxy, —OS(O)₂-alkyl,—OS(O)₂-substituted alkyl, —OS(O)₂-aryl, —OS(O)₂-substituted aryl,—OS(O)₂-heteroaryl, —OS(O)₂-substituted heteroaryl,—OS(O)₂-heterocyclic, —OS(O)₂-substituted heterocyclic, —OSO₂—NRR whereR is hydrogen or alkyl, —NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl,—NRS(O)₂-aryl, —NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl,—NRS(O)₂-substituted heteroaryl, —NRS(O)₂-heterocyclic,—NRS(O)₂-substituted heterocyclic, —NRS(O)₂—NR-alkyl,—NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl, —NRS(O)₂—NR-substitutedaryl, —NRS(O)₂—NR-heteroaryl, —NRS(O)₂—NR-substituted heteroaryl,—NRS(O)₂—NR-heterocyclic, —NRS(O)₂—NR-substituted heterocyclic where Ris hydrogen or alkyl, mono- and di-alkylamino, mono- and di-(substitutedalkyl)amino, mono- and di-arylamino, mono- and di-substituted arylamino,mono- and di-heteroarylamino, mono- and di-substituted heteroarylamino,mono- and di-heterocyclic amino, mono- and di-substituted heterocyclicamino, unsymmetric di-substituted amines having different substituentsindependently selected from the group consisting of alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic and substituted alkyl groupshaving amino groups blocked by conventional blocking groups such as Boc,Cbz, formyl, and the like or alkyl/substituted alkyl groups substitutedwith —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-alkenyl, —SO₂-substitutedalkenyl, —SO₂-cycloalkyl, —SO₂-substituted cycloalkyl, —SO₂-aryl,—SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substituted heteroaryl,—SO₂-heterocyclic, —SO₂-substituted heterocyclic and —SO₂NRR where R ishydrogen or alkyl.

Preferably, the substituents are independently selected from the groupconsisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminocarbonylamino,aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryloxy,carboxyl, carboxyl esters, cyano, cycloalkyl, substituted cycloalkyl,cycloalkyloxy, substituted cycloalkyloxy, halogen, heteroaryl,substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy,heterocyclic, substituted heterocyclic, hydroxyl, nitro, andoxycarbonylamino.

“Amidino” refers to the group H₂NC(═NH)— and the term “alkylamidino”refers to compounds having 1 to 3 alkyl groups (e.g., alkylHNC(═NH)—).

“Amino” refers to the group —NH₂.

“Substituted amino” refers to the group —NRR, where each R group isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-alkenyl,—SO₂-substituted alkenyl, —SO₂-cycloalkyl, —SO₂-substituted cycloalkyl,—SO₂-aryl, —SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substitutedheteroaryl, —SO₂-heterocyclic, —SO₂-substituted heterocyclic, providedthat both R groups are not hydrogen; or the R groups can be joinedtogether with the nitrogen atom to form a heterocyclic or substitutedheterocyclic ring.

“Aminoacyl” refers to the groups —NRC(O)alkyl, —NRC(O)substituted alkyl,—NRC(O)cycloalkyl, —NRC(O)substituted cycloalkyl, —NRC(O)alkenyl,—NRC(O)substituted alkenyl, —NRC(O)alkynyl, —NRC(O)substituted alkynyl,—NRC(O)aryl, —NRC(O)substituted aryl, —NRC(O)heteroaryl,—NRC(O)substituted heteroaryl, —NRC(O)heterocyclic, and—NRC(O)substituted heterocyclic where R is hydrogen or alkyl and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Aryl” or “Ar” refers to an unsaturated aromatic carbocyclic group offrom 6 to 14 carbon atoms having a single ring (e.g., phenyl) ormultiple condensed rings (e.g., naphthyl or anthryl) which condensedrings may or may not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7yl, and the like) provided that the pointof attachment is through an aromatic ring atom. Preferred aryls includephenyl, naphthyl and 5,6,7,8-tetrahydronaphth-2-yl.

“Substituted aryl” refers to aryl groups which are substituted with from1 to 3 substituents selected from the group consisting of hydroxy, acyl,acylamino, thiocarbonylamino, acyloxy, alkyl, substituted alkyl, alkoxy,substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, amidino, alkylamidino, thioamidino, amino, aminoacyl,aminocarbonyloxy, aminocarbonylamino, aminothiocarbonylamino, aryl,substituted aryl, aryloxy, substituted aryloxy, cycloalkoxy, substitutedcycloalkoxy, heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy,substituted heterocyclyloxy, carboxyl, carboxylalkyl,carboxyl-substituted alkyl, carboxyl-cycloalkyl, carboxyl-substitutedcycloalkyl, carboxylaryl, carboxyl-substituted aryl, carboxylheteroaryl,carboxyl-substituted heteroaryl, carboxylheterocyclic,carboxyl-substituted heterocyclic, carboxylamido, cyano, thiol,thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl,thioheteroaryl, substituted thioheteroaryl, thiocycloalkyl, substitutedthiocycloalkyl, thioheterocyclic, substituted thioheterocyclic,cycloalkyl, substituted cycloalkyl, guanidino, guanidinosulfone, halo,nitro, heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkoxy, substituted cycloalkoxy, heteroaryloxy,substituted heteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy,oxycarbonylamino, oxythiocarbonylamino, —S(O)₂-alkyl, —S(O)₂-substitutedalkyl, —S(O)₂-cycloalkyl, —S(O)₂-substituted cycloalkyl, —S(O)₂-alkenyl,—S(O)₂-substituted alkenyl, —S(O)₂-aryl, —S(O)₂-substituted aryl,—S(O)₂-heteroaryl, —S(O)₂-substituted heteroaryl, —S(O)₂-heterocyclic,—S(O)₂-substituted heterocyclic, —OS(O)₂-alkyl, —OS(O)₂-substitutedalkyl, —OS(O)₂-aryl, —OS(O)₂-substituted aryl, —OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂-heterocyclic,—OS(O)₂-substituted heterocyclic, —OSO₂—NRR where R is hydrogen oralkyl, —NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl,—NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl, —NRS(O)₂-substitutedheteroaryl, —NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl, —NRS(O)₂—NR—substituted heteroaryl, —NRS(O)₂—NR-heterocyclic,—NRS(O)₂—NR-substituted heterocyclic where R is hydrogen or alkyl, mono-and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- anddi-arylamino, mono- and di-substituted arylamino, mono- anddi-heteroarylamino, mono- and di-substituted heteroarylamino, mono- anddi-heterocyclic amino, mono- and di-substituted heterocyclic amino,unsymmetric di-substituted amines having different substituentsindependently selected from the group consisting of alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic and amino groups on thesubstituted aryl blocked by conventional blocking groups such as Boc,Cbz, formyl, and the like or substituted with —SO₂NRR where R ishydrogen or alkyl.

Preferred substituents are selected from the group consisting ofhydroxy, acyl, acylamino, acyloxy, alkyl, substituted alkyl, alkoxy,substituted alkoxy, alkenyl, substituted alkenyl, amino, substitutedamino, aminoacyl, aminocarbonyloxy, aminocarbonylamino, aryl,substituted aryl, aryloxy, substituted aryloxy, cycloalkoxy, substitutedcycloalkoxy, heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy,substituted heterocyclyloxy, carboxyl, carboxyl esters, cyano,cycloalkyl, substituted cycloalkyl, halo, nitro, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, and oxycarbonylamino

“Cycloalkenyl” refers to cyclic alkenyl groups of from 3 to 8 carbonatoms having single or multiple unsaturation but which are not aromatic.

“Cycloalkoxy” refers to —O-cycloalkyl groups.

“Substituted cycloalkoxy” refers to —O-substituted cycloalkyl groups.

“Cycloalkyl”, with regard to the compounds of Formulae I and II and thePEG derivatives, refers to cyclic alkyl groups of from 3 to 12 carbonatoms having a single or multiple condensed rings including, by way ofexample, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cyclooctyl and the like. Preferably “cycloalkyl” refers to cyclic alkylgroups of from 3 to 8 carbon atoms having a single cyclic ring.

“Cycloalkyl”, with regards to the compounds of Formulae III-IX, refersto cyclic alkyl groups of from 3 to 8 carbon atoms having a singlecyclic ring including, by way of example, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclooctyl and the like. Excluded from thisdefinition are multi-ring alkyl groups such as adamantanyl, etc.

“Lower cycloalkyl” refers to cyclic alkyl groups of from 3 to 6 carbonatoms having a single cyclic ring including, by way of example,cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

“Substituted cycloalkyl” and “substituted cycloalkenyl” refers to acycloalkyl or cycloalkenyl group, preferably of from 3 to 8 carbonatoms, having from 1 to 5 substituents independently selected from thegroup consisting of oxo (═O), thioxo (═S), alkoxy, substituted alkoxy,acyl, acylamino, thiocarbonylamino, acyloxy, amino, amidino,alkylamidino, thioamidino, aminoacyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aryl, substituted aryl,aryloxy, substituted aryloxy, aryloxyaryl, substituted aryloxyaryl,halogen, hydroxyl, cyano, nitro, carboxyl, carboxylalkyl,carboxyl-substituted alkyl, carboxyl-cycloalkyl, carboxyl-substitutedcycloalkyl, carboxylaryl, carboxyl-substituted aryl, carboxylheteroaryl,carboxyl-substituted heteroaryl, carboxylheterocyclic,carboxyl-substituted heterocyclic, cycloalkyl, substituted cycloalkyl,guanidino, guanidinosulfone, thiol, thioalkyl, substituted thioalkyl,thioaryl, substituted thioaryl, thiocycloalkyl, substitutedthiocycloalkyl, thioheteroaryl, substituted thioheteroaryl,thioheterocyclic, substituted thioheterocyclic, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy,substituted cycloalkoxy, heteroaryloxy, substituted heteroaryloxy,heterocyclyloxy, substituted heterocyclyloxy, oxycarbonylamino,oxythiocarbonylamino, —OS(O)₂-alkyl, —OS(O)₂-substituted alkyl,—OS(O)₂-aryl, —OS(O)₂-substituted aryl, —OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂-heterocyclic,—OS(O)₂-substituted heterocyclic, —OSO₂—NRR where R is hydrogen oralkyl, —NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl,—NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl, —NRS(O)₂-substitutedheteroaryl, —NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR— substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-heterocyclic,—NRS(O)₂—NR-substituted heterocyclic where R is hydrogen or alkyl, mono-and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- anddi-arylamino, mono- and di-substituted arylamino, mono- anddi-heteroarylamino, mono- and di-substituted heteroarylamino, mono- anddi-heterocyclic amino, mono- and di-substituted heterocyclic amino,unsymmetric di-substituted amines having different substituentsindependently selected from the group consisting of alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic and substituted alkynyl groupshaving amino groups blocked by conventional blocking groups such as Boc,Cbz, formyl, and the like or alkynyl/substituted alkynyl groupssubstituted with —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-alkenyl,—SO₂-substituted alkenyl, —SO₂-cycloalkyl, —SO₂-substituted cycloalkyl,—SO₂-aryl, —SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substitutedheteroaryl, —SO₂-heterocyclic, —SO₂-substituted heterocyclic and —SO₂NRRwhere R is hydrogen or alkyl.

Preferred substituents are selected from the group consisting of oxo(═O), thioxo (═S), alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminocarbonylamino,aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryloxy,carboxyl, carboxyl esters, cyano, cycloalkyl, substituted cycloalkyl,cycloalkyloxy, substituted cycloalkyloxy, halogen, heteroaryl,substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy,heterocyclic, substituted heterocyclic, hydroxyl, nitro, andoxycarbonylamino.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo andpreferably is fluoro, chloro or bromo.

“Heteroaryl” refers to an aromatic carbocyclic group of from 2 to 10carbon atoms and 1 to 4 heteroatoms selected from the group consistingof oxygen, nitrogen and sulfur within the ring or oxides thereof. Suchheteroaryl groups can have a single ring (e.g., pyridyl or furyl) ormultiple condensed rings (e.g., indolizinyl or benzothienyl) wherein oneor more of the condensed rings may or may not be aromatic provided thatthe point of attachment is through an aromatic ring atom. Additionally,the heteroatoms of the heteroaryl group may be oxidized, i.e., to formpyridine N-oxides or 1,1-dioxo-1,2,5-thiadiazoles and the like.Additionally, the carbon atoms of the ring may be substituted with anoxo (═O). Preferred heteroaryls include pyridyl, pyrrolyl, indolyl,furyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1-oxo-1,2,5-thiadiazolyl and1,1-dioxo-1,2,5-thiadiazolyl. The term “heteroaryl having two nitrogenatoms in the heteroaryl ring” refers to a heteroaryl group having two,and only two, nitrogen atoms in the heteroaryl ring and optionallycontaining 1 or 2 other heteroatoms in the heteroaryl ring, such asoxygen or sulfur.

“Substituted heteroaryl” refers to heteroaryl groups which aresubstituted with from 1 to 3 substituents selected from the groupconsisting of hydroxy, acyl, acylamino, thiocarbonylamino, acyloxy,alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, amidino,alkylamidino, thioamidino, amino, aminoacyl, aminocarbonyloxy,aminocarbonylamino, aminothiocarbonylamino, aryl, substituted aryl,aryloxy, substituted aryloxy, cycloalkoxy, substituted cycloalkoxy,heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy, substitutedheterocyclyloxy, carboxyl, carboxylalkyl, carboxyl-substituted alkyl,carboxyl-cycloalkyl, carboxyl-substituted cycloalkyl, carboxylaryl,carboxyl-substituted aryl, carboxylheteroaryl, carboxyl-substitutedheteroaryl, carboxylheterocyclic, carboxyl-substituted heterocyclic,carboxylamido, cyano, thiol, thioalkyl, substituted thioalkyl, thioaryl,substituted thioaryl, thioheteroaryl, substituted thioheteroaryl,thiocycloalkyl, substituted thiocycloalkyl, thioheterocyclic,substituted thioheterocyclic, cycloalkyl, substituted cycloalkyl,guanidino, guanidinosulfone, halo, nitro, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy,substituted cycloalkoxy, heteroaryloxy, substituted heteroaryloxy,heterocyclyloxy, substituted heterocyclyloxy, oxycarbonylamino,oxythiocarbonylamino, —S(O)₂-alkyl, —S(O)₂-substituted alkyl,—S(O)₂-cycloalkyl, —S(O)₂-substituted cycloalkyl, —S(O)₂-alkenyl,—S(O)₂-substituted alkenyl, —S(O)₂-aryl, —S(O)₂-substituted aryl,—S(O)₂-heteroaryl, —S(O)₂-substituted heteroaryl, —S(O)₂-heterocyclic,—S(O)₂-substituted heterocyclic, —OS(O)₂-alkyl, —OS(O)₂-substitutedalkyl, —OS(O)₂-aryl, —OS(O)₂-substituted aryl, —OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂-heterocyclic,—OS(O)₂-substituted heterocyclic, —OSO₂—NRR where R is hydrogen oralkyl, —NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl,—NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl, —NRS(O)₂-substitutedheteroaryl, —NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl, —NRS(O)₂—NR—substituted heteroaryl, —NRS(O)₂—NR-heterocyclic,—NRS(O)₂—NR-substituted heterocyclic where R is hydrogen or alkyl, mono-and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- anddi-arylamino, mono- and di-substituted arylamino, mono- anddi-heteroarylamino, mono- and di-substituted heteroarylamino, mono- anddi-heterocyclic amino, mono- and di-substituted heterocyclic amino,unsymmetric di-substituted amines having different substituentsindependently selected from the group consisting of alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic and amino groups on thesubstituted aryl blocked by conventional blocking groups such as Boc,Cbz, formyl, and the like or substituted with —SO₂NRR where R ishydrogen or alkyl.

Preferably the substituents are selected from the group consisting ofthose defined above as preferred for substituted aryl.

“Heteroaryloxy” refers to the group —O-heteroaryl and “substitutedheteroaryloxy” refers to the group —O-substituted heteroaryl.

“Heteroaralkoxy” refers to the group heteroaryl-alkylene-O—.

“Substituted heteroaralkoxy” refers to the group substitutedheteroaryl-alkylene-O—.

“Heterocycle” or “heterocyclic” refers to a saturated or unsaturatedgroup having a single ring or multiple condensed rings, from 1 to 10carbon atoms and from 1 to 4 hetero atoms selected from the groupconsisting of nitrogen, sulfur or oxygen within the ring wherein, infused ring systems, one or more the rings can be aryl or heteroaryl.

“Substituted heterocyclic” refers to heterocycle groups which aresubstituted with from 1 to 3 substituents selected from the groupconsisting of oxo (═O), thioxo (═S), alkoxy, substituted alkoxy, acyl,acylamino, thiocarbonylamino, acyloxy, amino, amidino, alkylamidino,thioamidino, aminoacyl, aminocarbonylamino, aminothiocarbonylamino,aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryloxy,aryloxyaryl, substituted aryloxyaryl, halogen, hydroxyl, cyano, nitro,carboxyl, carboxylalkyl, carboxyl-substituted alkyl,carboxyl-cycloalkyl, carboxyl-substituted cycloalkyl, carboxylaryl,carboxyl-substituted aryl, carboxylheteroaryl, carboxyl-substitutedheteroaryl, carboxylheterocyclic, carboxyl-substituted heterocyclic,cycloalkyl, substituted cycloalkyl, guanidino, guanidinosulfone, thiol,thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl,thiocycloalkyl, substituted thiocycloalkyl, thioheteroaryl, substitutedthioheteroaryl, thioheterocyclic, substituted thioheterocyclic,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkoxy, substituted cycloalkoxy, heteroaryloxy,substituted heteroaryloxy, —C(O)O-aryl, —C(O)O-substituted aryl,heterocyclyloxy, substituted heterocyclyloxy, oxycarbonylamino,oxythiocarbonylamino, —OS(O)₂-alkyl, —OS(O)₂-substituted alkyl,—OS(O)₂-aryl, —OS(O)₂-substituted aryl, —OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂-heterocyclic,—OS(O)₂-substituted heterocyclic, —OSO₂—NRR where R is hydrogen oralkyl, —NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl,—NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl, —NRS(O)₂-substitutedheteroaryl, —NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR— substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-heterocyclic,—NRS(O)₂—NR-substituted heterocyclic where R is hydrogen or alkyl, mono-and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- anddi-arylamino, mono- and di-substituted arylamino, mono- anddi-heteroarylamino, mono- and di-substituted heteroarylamino, mono- anddi-heterocyclic amino, mono- and di-substituted heterocyclic amino,unsymmetric di-substituted amines having different substituentsindependently selected from the group consisting of alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic and substituted alkynyl groupshaving amino groups blocked by conventional blocking groups such as Boc,Cbz, formyl, and the like or alkynyl/substituted alkynyl groupssubstituted with —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-alkenyl,—SO₂-substituted alkenyl, —SO₂-cycloalkyl, —SO₂-substituted cycloalkyl,—SO₂-aryl, —SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substitutedheteroaryl, —SO₂-heterocyclic, —SO₂-substituted heterocyclic and —SO₂NRRwhere R is hydrogen or alkyl.

Preferably, the substituents are selected from the group consisting ofthe preferred substitutents defined for substituted cycloalkyl.

Examples of heterocycles and heteroaryls include, but are not limitedto, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholino, morpholinyl, thiomorpholino,thiomorpholinyl (also referred to as thiamorpholinyl), piperidinyl,pyrrolidine, tetrahydrofuranyl, and the like.

“Optionally substituted” means that the recited group may beunsubstituted or the recited group may be substituted.

“Pharmaceutically acceptable carrier” means a carrier that is useful inpreparing a pharmaceutical composition or formulation that is generallysafe, non-toxic and neither biologically nor otherwise undesirable, andincludes a carrier that is acceptable for veterinary use as well ashuman pharmaceutical use. A pharmaceutically acceptable carrier orexcipient as used in the specification and claims includes both one ormore than one of such carriers.

“Pharmaceutically-acceptable cation” refers to the cation of apharmaceutically-acceptable salt.

“Pharmaceutically acceptable salt” refers to salts which retain thebiological effectiveness and properties of the compounds of thisinvention and which are not biologically or otherwise undesirable.Pharmaceutically acceptable salts refer to pharmaceutically acceptablesalts of the compounds, which salts are derived from a variety oforganic and inorganic counter ions well known in the art and include, byway of example only, sodium, potassium, calcium, magnesium, ammonium,tetraalkylammonium, and the like; and when the molecule contains a basicfunctionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate,oxalate and the like.

Pharmaceutically-acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl)amines, tri(substituted alkyl)amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl)amines, tri(substituted alkenyl)amines,cycloalkyl amines, di(cycloalkyl)amines, tri(cycloalkyl)amines,substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines,di(cycloalkenyl)amines, tri(cycloalkenyl)amines, substitutedcycloalkenyl amines, disubstituted cycloalkenyl amine, trisubstitutedcycloalkenyl amines, aryl amines, diaryl amines, triaryl amines,heteroaryl amines, diheteroaryl amines, triheteroaryl amines,heterocyclic amines, diheterocyclic amines, triheterocyclic amines,mixed di- and tri-amines where at least two of the substituents on theamine are different and are selected from the group consisting of alkyl,substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl,heterocyclic, and the like. Also included are amines where the two orthree substituents, together with the amino nitrogen, form aheterocyclic or heteroaryl group.

Examples of suitable amines include, by way of example only,isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl)amine,tri(n-propyl)amine, ethanolamine, 2-dimethylaminoethanol, tromethamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine,purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and thelike.

It should also be understood that other carboxylic acid derivativeswould be useful in the practice of this invention, for example,carboxylic acid amides, including carboxamides, lower alkylcarboxamides, dialkyl carboxamides, and the like.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

A compound of Formula (I) may act as a pro-drug. Prodrug means anycompound which releases an active parent drug according to Formula (I)in vivo when such prodrug is administered to a mammalian subject.Prodrugs of a compound of Formula (I) are prepared by modifyingfunctional groups present in the compound of Formula (I) in such a waythat the modifications may be cleaved in vivo to release the parentcompound. Prodrugs include compounds of Formula (I) wherein a hydroxy,amino, or sulfhydryl group in compound (I) is bonded to any group thatmay be cleaved in vivo to regenerate the free hydroxyl, amino, orsulfhydryl group, respectively. Examples of prodrugs include, but arenot limited to esters (e.g., acetate, formate, and benzoatederivatives), carbamates (e.g., N,N-dimethylamino-carbonyl) of hydroxyfunctional groups in compounds of Formula (I), and the like.

“Treating” or “treatment” of a disease includes:

-   -   (1) preventing the disease, i.e. causing the clinical symptoms        of the disease not to develop in a mammal that may be exposed to        or predisposed to the disease but does not yet experience or        display symptoms of the disease,    -   (2) inhibiting the disease, i.e., arresting or reducing the        development of the disease or its clinical symptoms, or    -   (3) relieving the disease, i.e., causing regression of the        disease or its clinical symptoms.

A “therapeutically effective amount” means the amount of a compound orantibody that, when administered to a mammal for treating a disease, issufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity and the age, weight, etc., of the mammal tobe treated.

Pharmaceutical Formulations of the Compounds

In general, the compounds of the subject invention will be administeredin a therapeutically effective amount by any of the accepted modes ofadministration for these compounds. The compounds can be administered bya variety of routes, including, but not limited to, oral, parenteral(e.g., subcutaneous, subdural, intravenous, intramuscular, intrathecal,intraperitoneal, intracerebral, intraarterial, or intralesional routesof administration), topical, intranasal, localized (e.g., surgicalapplication or surgical suppository), rectal, and pulmonary (e.g.,aerosols, inhalation, or powder). Accordingly, these compounds areeffective as both injectable and oral compositions. The compounds can beadministered continuously by infusion or by bolus injection. Preferably,the compounds are administered by parenteral routes. More preferably,the compounds are administered by intravenous routes. Such compositionsare prepared in a manner well known in the pharmaceutical art.

The actual amount of the compound of the subject invention, i.e., theactive ingredient, will depend on a number of factors, such as theseverity of the disease, i.e., the condition or disease to be treated,age and relative health of the subject, the potency of the compoundused, the route and form of administration, and other factors.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range which includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography. Theeffective blood level of the compounds of the subject invention ispreferably greater than or equal to 10 ng/ml.

The amount of the pharmaceutical composition administered to the patientwill vary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like, hi therapeuticapplications, compositions are administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications. Anamount adequate to accomplish this is defined as “therapeuticallyeffective dose.” Amounts effective for this use will depend on thedisease condition being treated as well as by the judgment of theattending clinician depending upon factors such as the severity of theinflammation, the age, weight and general condition of the patient, andthe like.

The compositions administered to a patient are in the form ofpharmaceutical compositions described supra. These compositions may besterilized by conventional sterilization techniques, or may be sterilefiltered. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile aqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The active compound is effective over a wide dosage range and isgenerally administered in a pharmaceutically or therapeuticallyeffective amount. The therapeutic dosage of the compounds of the presentinvention will vary according to, for example, the particular use forwhich the treatment is made, the manner of administration of thecompound, the health and condition of the patient, and the judgment ofthe prescribing physician. For example, for intravenous administration,the dose will typically be in the range of about 0.5 mg to about 100 mgper kilogram body weight, preferably about 3 mg to about 50 mg perkilogram body weight. Effective doses can be extrapolated fromdose-response curves derived from in vitro or animal model test systems.Typically, the clinician will administer the compound until a dosage isreached that achieves the desired effect.

When employed as pharmaceuticals, the compounds of the subject inventionare usually administered in the form of pharmaceutical compositions.This invention also includes pharmaceutical compositions, which containas the active ingredient, one or more of the compounds of the subjectinvention above, associated with one or more pharmaceutically acceptablecarriers or excipients. The excipient employed is typically one suitablefor administration to human subjects or other mammals. In making thecompositions of this invention, the active ingredient is usually mixedwith an excipient, diluted by an excipient or enclosed within a carrierwhich can be in the form of a capsule, sachet, paper or other container.When the excipient serves as a diluent, it can be a solid, semi-solid,or liquid material, which acts as a vehicle, carrier or medium for theactive ingredient. Thus, the compositions can be in the form of tablets,pills, powders, lozenges, sachets, cachets, elixirs, suspensions,emulsions, solutions, syrups, aerosols (as a solid or in a liquidmedium), ointments containing, for example, up to 10% by weight of theactive compound, soft and hard gelatin capsules, suppositories, sterileinjectable solutions, and sterile packaged powders.

In preparing a formulation, it may be necessary to mill the activecompound to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound is substantiallyinsoluble, it ordinarily is milled to a particle size of less than 200mesh. If the active compound is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g., about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The quantity of active compound in the pharmaceutical composition andunit dosage form thereof may be varied or adjusted widely depending uponthe particular application, the manner or introduction, the potency ofthe particular compound, and the desired concentration. The term “unitdosage forms” refers to physically discrete units suitable as unitarydosages for human subjects and other mammals, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical excipient. The concentration of therapeutically activecompound may vary from about 1 mg/ml to 250 g/ml.

Preferably, the compound can be formulated for parenteral administrationin a suitable inert carrier, such as a sterile physiological salinesolution. For example, the concentration of compound in the carriersolution is typically between about 1-100 mg/ml. The dose administeredwill be determined by route of administration. Preferred routes ofadministration include parenteral or intravenous administration. Atherapeutically effective dose is a dose effective to produce asignificant steroid tapering. Preferably, the amount is sufficient toproduce a statistically significant amount of steroid tapering in asubject.

Administration of therapeutic agents by intravenous formulation is wellknown in the pharmaceutical industry. An intravenous formulation shouldpossess certain qualities aside from being just a composition in whichthe therapeutic agent is soluble. For example, the formulation shouldpromote the overall stability of the active ingredient(s), also, themanufacture of the formulation should be cost effective. All of thesefactors ultimately determine the overall success and usefulness of anintravenous formulation.

Other accessory additives that may be included in pharmaceuticalformulations of compounds of the present invention as follow: solvents:ethanol, glycerol, propylene glycol; stabilizers: EDTA (ethylene diaminetetraacetic acid), citric acid; antimicrobial preservatives: benzylalcohol, methyl paraben, propyl paraben; buffering agents: citricacid/sodium citrate, potassium hydrogen tartrate, sodium hydrogentartrate, acetic acid/sodium acetate, maleic acid/sodium maleate, sodiumhydrogen phthalate, phosphoric acid/potassium dihydrogen phosphate,phosphoric acid/disodium hydrogen phosphate; and tonicity modifiers:sodium chloride, mannitol, dextrose.

The presence of a buffer is necessary to maintain the aqueous pH in therange of from about 4 to about 8 and more preferably in a range of fromabout 4 to about 6. The buffer system is generally a mixture of a weakacid and a soluble salt thereof, e.g., sodium citrate/citric acid; orthe monocation or dication salt of a dibasic acid, e.g., potassiumhydrogen tartrate; sodium hydrogen tartrate, phosphoric acid/potassiumdihydrogen phosphate, and phosphoric acid/disodium hydrogen phosphate.

The amount of buffer system used is dependent on (1) the desired pH; and(2) the amount of drug. Generally, the amount of buffer used is in a0.5:1 to 50:1 mole ratio of buffenalendronate (where the moles of bufferare taken as the combined moles of the buffer ingredients, e.g., sodiumcitrate and citric acid) of formulation to maintain a pH in the range of4 to 8 and generally, a 1:1 to 10:1 mole ratio of buffer (combined) todrug present is used.

A useful buffer in the invention is sodium citrate/citric acid in therange of 5 to 50 mg per ml. sodium citrate to 1 to 15 mg per ml. citricacid, sufficient to maintain an aqueous pH of 4-6 of the composition.

The buffer agent may also be present to prevent the precipitation of thedrug through soluble metal complex formation with dissolved metal ions,e.g., Ca, Mg, Fe, Al, Ba, which may leach out of glass containers orrubber stoppers or be present in ordinary tap water. The agent may actas a competitive complexing agent with the drug and produce a solublemetal complex leading to the presence of undesirable particulates.

In addition, the presence of an agent, e.g., sodium chloride in anamount of about of 1-8 mg/ml, to adjust the tonicity to the same valueof human blood may be required to avoid the swelling or shrinkage oferythrocytes upon administration of the intravenous formulation leadingto undesirable side effects such as nausea or diarrhea and possibly toassociated blood disorders. In general, the tonicity of the formulationmatches that of human blood which is in the range of 282 to 288 mOsm/kg,and in general is 285 mOsm/kg, which is equivalent to the osmoticpressure corresponding to a 0.9% solution of sodium chloride.

The intravenous formulation can be administered by direct intravenousinjection, i.v. bolus, or can be administered by infusion by addition toan appropriate infusion solution such as 0.9% sodium chloride injectionor other compatible infusion solution.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 5 to about 100 mg, more usually about 10 toabout 30 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

The active compound is effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It, willbe understood, however, that the amount of the compound actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the activeingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. Preferably the compositions are administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be breathed directly from thenebulizing device or the nebulizing device may be attached to a facemasks tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices which deliver the formulationin an appropriate manner.

The compounds of this invention can be administered in a sustainedrelease form. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe protein, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981)and Langer, Chem. Tech. 12: 98-105 (1982) or polyvinyl alcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand gamma ethyl-L-glutamate (Sidman et al., Biopolymers 22: 547-556,1983), non-degradable ethylene-vinyl acetate (Langer et ah, supra),degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (i.e., injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

The compounds of this invention can be administered in a sustainedrelease form, for example a depot injection, implant preparation, orosmotic pump, which can be formulated in such a manner as to permit asustained release of the active ingredient. Implants for sustainedrelease formulations are well-known in the art. Implants may beformulated as, including but not limited to, microspheres, slabs, withbiodegradable or non-biodegradable polymers. For example, polymers oflactic acid and/or glycolic acid form an erodible polymer that iswell-tolerated by the host. The implant is placed in proximity to thesite of protein deposits (e.g., the site of formation of amyloiddeposits associated with neurodegenerative disorders), so that the localconcentration of active agent is increased at that site relative to therest of the body.

The following formulation examples illustrate pharmaceuticalcompositions of the present invention.

Formulation Example 1

Hard gelatin capsules containing the following ingredients are prepared:

Quantity Ingredient (mg/capsule) Active Ingredient 30.0 Starch 305.0Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in340 mg quantities.

Formulation Example 2

A tablet formula is prepared using the ingredients below:

Quantity Ingredient (mg/capsule) Active Ingredient 25.0 Cellulose,microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

The components are blended and compressed to form tablets, each weighing240 mg.

Formulation Example 3

A dry powder inhaler formulation is prepared containing the followingcomponents:

Ingredient Weight % Active Ingredient 5 Lactose 95

The active mixture is mixed with the lactose and the mixture is added toa dry powder inhaling appliance.

Formulation Example 4

Tablets, each containing 30 mg of active ingredient, are prepared asfollows:

Quantity Ingredient (mg/capsule) Active Ingredient 30.0 mg Starch 45.0mg Microcrystalline cellulose 35.0 mg Polyvinylpyrrolidone 4.0 mg (as10% solution in water) Sodium Carboxymethyl starch 4.5 mg Magnesiumstearate 0.5 mg Talc 1.0 mg Total 120 mg

The active ingredient, starch and cellulose are passed through a No. 20mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinyl-pyrrolidone is mixed with the resultant powders, which arethen passed through a 16 mesh U.S. sieve. The granules so produced aredried at 50° to 60° C. and passed through a 16 mesh U.S. sieve. Thesodium carboxymethyl starch, magnesium stearate, and talc, previouslypassed through a No. 30 mesh U.S. sieve, are then added to the granules,which after mixing, are compressed on a tablet machine to yield tabletseach weighing 150 mg.

Formulation Example 5 Capsules, Each Containing 40 Mg of Medicament areMade as Follows

Quantity Ingredient (mg/capsule) Active Ingredient 40.0 mg Starch 109.0mg Magnesium stearate 1.0 mg Total 150.0 mg

The active ingredient, cellulose, starch, an magnesium stearate areblended, passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 150 mg quantities.

Formulation Example 6 Suppositories, Each Containing 25 Mg of ActiveIngredient are Made as Follows

Ingredient Amount Active Ingredient 25 mg Saturated fatty acidsglycerides to 2,000 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

Formulation Example 7 Suspensions, Each Containing 50 Mg of MedicamentPer 5.0 Ml Dose are Made as Follows

Ingredient Amount Active Ingredient 50.0 mg Xanthan gum 4.0 mg Sodiumcarboxymethyl cellose (11%) 500 mg Microcrystalline cellulose (89%)Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and color q.v. Purifiedwater to 5.0 ml

The medicament, sucrose and xanthan gum are blended, passed through aNo. 10 mesh U.S. sieve, and then mixed with a previously made solutionof the microcrystalline cellulose and sodium carboxymethyl cellulose inwater. The sodium benzoate, flavor, and color are diluted with some ofthe water and added with stirring. Sufficient water is then added toproduce the required volume.

Formulation Example 8

Hard gelatin tablets, each containing 15 mg of active ingredient aremade as follows:

Quantity Ingredient (mg/capsule) Active Ingredient 15.0 mg Starch 407.0mg Magnesium stearate 3.0 mg Total 425.0 mg

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 560 mg quantities.

Formulation Example 9 An Intravenous Formulation May be Prepared asFollows

Ingredient Quantity Active Ingredient 250.0 mg Isotonic saline 1000 ml

Therapeutic compound compositions generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle orsimilar sharp instrument.

Formulation Example 10 A Topical Formulation May be Prepared as Follows

Ingredient Quantity Active Ingredient 1-10 g Emulsifying Wax 30 g LiquidParaffin 20 g White Soft Paraffin to 100 g

The white soft paraffin is heated until molten. The liquid paraffin andemulsifying wax are incorporated and stirred until dissolved. The activeingredient is added and stirring is continued until dispersed. Themixture is then cooled until solid.

Formulation Example 11

An aerosol formulation may be prepared as follows: A solution of thecandidate compound in 0.5% sodium bicarbonate/saline (w/v) at aconcentration of 30.0 mg/mL is prepared using the following procedure:

A. Preparation of 0.5% Sodium Bicarbonate/Saline Stock Solution: 100.0mL

Ingredient Gram/100.0 mL Final Concentration Sodium Bicarbonate 0.5 g0.5% Saline q.s. ad 100.0 mL q.s. ad 100%

Procedure:

-   -   1. Add 0.5 g sodium bicarbonate into a 100 mL volumetric flask.    -   2. Add approximately 90.0 mL saline and sonicate until        dissolved.    -   3. Q.S. to 100.0 mL with saline and mix thoroughly.

B. Preparation of 30.0 Mg/mL Candidate Compound: 10.0 mL

Ingredient Gram/10.0 mL Final Concentration Candidate Compound 0.300 g30.0 mg/mL 0.5% Sodium Bicarbonate/ q.s. ad 10.0 mL q.s ad 100% SalineStock Solution

Procedure:

-   -   1. Add 0.300 g of the candidate compound into a 10.0 mL        volumetric flask.    -   2. Add approximately 9.7 mL of 0.5% sodium bicarbonate/saline        stock solution.    -   3. Sonicate until the candidate compound is completely        dissolved.    -   4. Q.S. to 10.0 mL with 0.5% sodium bicarbonate/saline stock        solution and mix

Another preferred formulation employed in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, herein incorporated byreference. Such patches may be constructed for continuous, pulsatile, oron demand delivery of pharmaceutical agents.

Direct or indirect placement techniques may be used when it is desirableor necessary to introduce the pharmaceutical composition to the brain.Direct techniques usually involve placement of a drug delivery catheterinto the host's ventricular system to bypass the blood-brain barrier.One such implantable delivery system used for the transport ofbiological factors to specific anatomical regions of the body isdescribed in U.S. Pat. No. 5,011,472, which is herein incorporated byreference.

Indirect techniques, which are generally preferred, usually involveformulating the compositions to provide for drug latentiation by theconversion of hydrophilic drugs into lipid-soluble drugs. Latentiationis generally achieved through blocking of the hydroxy, carbonyl,sulfate, and primary amine groups present on the drug to render the drugmore lipid soluble and amenable to transportation across the blood-brainbarrier. Alternatively, the delivery of hydrophilic drugs may beenhanced by intra-arterial infusion of hypertonic solutions which cantransiently open the blood-brain barrier.

In order to enhance serum half-life, the compounds may be encapsulated,introduced into the lumen of liposomes, prepared as a colloid, or otherconventional techniques may be employed which provide an extended serumhalf-life of the compounds. A variety of methods are available forpreparing liposomes, as described in, e.g., Szoka et al., U.S. Pat. Nos.4,235,871, 4,501,728 and 4,837,028 each of which is incorporated hereinby reference.

Pharmaceutical compositions of the invention are suitable for use in avariety of drug delivery systems. Suitable formulations for use in thepresent invention are found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985).

Utility

The compounds and pharmaceutical compositions of the invention showbiological activity in treating and preventing viral infections andassociated diseases, and, accordingly, have utility in treating viralinfections and associated diseases, such as Hemorrhagic fever viruses,in mammals including humans.

As noted above, the compounds described herein are suitable for use in avariety of drug delivery systems described above. Additionally, in orderto enhance the in vivo serum half life of the administered compound, thecompounds may be encapsulated, introduced into the lumen of liposomes,prepared as a colloid, or other conventional techniques may be employedwhich provide an extended serum half life of the compounds. A variety ofmethods are available for preparing liposomes, as described in, e.g.,Szoka, et ah, U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028, each ofwhich is incorporated herein by reference.

The amount of compound administered to the patient will vary dependingupon what is being administered, the purpose of the administration, suchas prophylaxis or therapy, the state of the patient, the manner ofadministration, and the like. In therapeutic applications, compositionsare administered to a patient already suffering from AD in an amountsufficient to at least partially arrest further onset of the symptoms ofthe disease and its complications. An amount adequate to accomplish thisis defined as “therapeutically effective dose.” Amounts effective forthis use will depend on the judgment of the attending cliniciandepending upon factors such as the degree or severity of AD in thepatient, the age, weight and general condition of the patient, and thelike. Preferably, for use as therapeutics, the compounds describedherein are administered at dosages ranging from about 0.1 to about 500mg/kg/day.

In prophylactic applications, compositions are administered to a patientat risk of developing AD (determined for example by genetic screening orfamilial trait) in an amount sufficient to inhibit the onset of symptomsof the disease. An amount adequate to accomplish this is defined as“prophylactically effective dose.” Amounts effective for this use willdepend on the judgment of the attending clinician depending upon factorssuch as the age, weight and general condition of the patient, and thelike. Preferably, for use as prophylactics, the compounds describedherein are administered at dosages ranging from about 0.1 to about 500mg/kg/day.

As noted above, the compounds administered to a patient are in the formof pharmaceutical compositions described above. These compositions maybe sterilized by conventional sterilization techniques, or may besterile filtered. When aqueous solutions are employed, these may bepackaged for use as is, or lyophilized, the lyophilized preparationbeing combined with a sterile aqueous carrier prior to administration.The pH of the compound preparations typically will be between 3 and 11,more preferably from 5-9 and most preferably from 7 and 8. It will beunderstood that use of certain of the foregoing excipients, carriers, orstabilizers will result in the formation of pharmaceutical salts.

Hemorrhagic fever viruses (HFVs) are RNA viruses that cause a variety ofdisease syndromes with similar clinical characteristics. HFVs that areof concern as potential biological weapons include but are not limitedto: Arenaviridae (Junin, Machupo, Guanavito, Sabia and Lassa),Filoviridae (ebola and Marburg viruses), Flaviviridae (yellow fever,omsk hemorrhagic fever and Kyasanur Forest disease viruses), andBunyaviridae (Rift Valley fever). The naturally occurring arenavirusesand potential engineered arenaviruses are included in the Category APathogen list according to the Center for Disease control and Preventionas being among those agents that have greatest potential for masscasualties.

Risk factors include: travel to Africa or Asia, handling of animalcarcasses, contact with infected animals or people, and/or arthropodbites. Arenaviruses are highly infectious after direct contact withinfected blood and/or bodily secretions. Humans usually become infectedthrough contact with infected rodents, the bite of an infectedarthropod, direct contact with animal carcasses, inhalation ofinfectious rodent excreta and/or injection of food contaminated withrodent excreta. The Tacaribe virus has been associated with bats.Airborne transmission of hemorrhagic fever is another mode, but somewhatless common Person-to-person contact may also occur in some cases.

All of the hemorrhagic fevers exhibit similar clinical symptoms.However, in general the clinical manifestations are non-specific andvariable. The incubation period is approximately 7-14 days. The onset isgradual with fever and malaise, tachypnea, relative bradycardia,hypotension, circulatory shock, conjeunctival injection, pharyngitis,lymphadenopathy, encephalitis, myalgia, back pain, headache anddizziness, as well as hyperesthesia of the skin. Some infected patientsmay not develop hemorrhagic manifestations.

Methods of diagnosis at specialized laboratories include antigendetection by antigen-capture enzyme-linked immunosorbent assay (ELISA),IgM antibody detection by antibody-capture enzyme-linked immunosorbentassay, reverse transcriptase polymerase chain reaction (RT-PCR), andviral isolation. Antigen detection (by enzyme-linked immunosorbentassay) and reverse transcriptase polymerase chain reaction are the mostuseful diagnostic techniques in the acute clinical setting. Viralisolation is of limited value because it requires a biosafety level 4(BSL-4) laboratory.

The present invention also provides for kits for detecting the presenceof arenavirus in a biological sample obtained from a subject usingST-375, the dansyl analogue of ST-294. The containers of the kit willgenerally include at least one vial, test tube, flask, bottle, syringeor other container means. The kits may also comprise a second/thirdcontainer means for containing a sterile, pharmaceutically acceptablebuffer or other diluent. The kits of the present invention will alsotypically include a means for containing the vials, or such like, andother component, in close confinement for commercial sale, such as,e.g., injection or blow-molded plastic containers into which the desiredvials and other apparatus are placed and retained.

In one aspect of the present invention, the kits include the means andinstructions for using ST-375 to detect and quantitate the presence ofan arenavirus in the biological sample. In one embodiment, thearenavirus is resistant to known drugs.

In another aspect of the present invention, the kits include the meansand instructions for using ST-375 to detect hit/leads for drugoptimization, in yet another aspect of the instant invention, the kitsinclude the means and instructions for using ST-375 for rapidlydetermining concentrations of various inhibitors or drugs in the serum.

The following synthetic and biological examples are offered toillustrate this invention and are not to be construed in any way aslimiting the scope of this invention.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and is not intended to limit thescope of what the inventors regard as their invention nor is it intendedto represent that the experiments below are all or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is weight averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Synthesis of Compounds

The compounds of formula I, as well as IA and IB above are readilyprepared via several divergent synthetic routes with the particularroute selected relative to the ease of compound preparation, thecommercial availability of starting materials, and the like.

The compounds of Formulae I and II can be prepared from readilyavailable starting materials using the following general methods andprocedures. It will be appreciated that where typical or preferredprocess conditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. Suitableprotecting groups for various functional groups as well as suitableconditions for protecting and deprotecting particular functional groupsare well known in the art. For example, numerous protecting groups aredescribed in T. W. Greene and G. M. Wuts, Protecting Groups in OrganicSynthesis, Second Edition, Wiley, New York, 1991, and references citedtherein.

Furthermore, the compounds of this invention will typically contain oneor more chiral centers. Accordingly, if desired, such compounds can beprepared or isolated as pure stereoisomers, i. e., as individualenantiomers or diastereomers, or as stereoisomer-enriched mixtures. Allsuch stereoisomers (and enriched mixtures) are included within the scopeof this invention, unless otherwise indicated. Pure stereoisomers (orenriched mixtures) may be prepared using, for example, optically activestarting materials or stereoselective reagents well-known in the art.Alternatively, racemic mixtures of such compounds can be separatedusing, for example, chiral column chromatography, chiral resolvingagents and the like.

Unless otherwise indicated, the products of this invention are a mixtureof R, S enantiomers. Preferably, however, when a chiral product isdesired, the chiral product can be obtained via purification techniqueswhich separates enantiomers from a R, S mixture to provide for one orthe other stereoisomer. Such techniques are known in the art.

In another embodiment, the compounds can be provided as prodrugs whichconvert (e.g., hydrolyze, metabolize, etc.) in vivo to a compound ofFormula I above. In a preferred example of such an embodiment, thecarboxylic acid group of the compound of Formula I is modified into agroup which, in vivo, will convert to a carboxylic acid group (includingsalts thereof).

In the examples below, if an abbreviation is not defined above, it hasits generally accepted meaning. Further, all temperatures are in degreesCelsius (unless otherwise indicated). The following Methods were used toprepare the compounds set forth below as indicated.

The following examples are provided to describe the invention in furtherdetail. These examples illustrate suitable methods for the synthesis ofrepresentative members of this invention. However, the methods ofsynthesis are intended to illustrate and not to limit the invention tothose exemplified below. The starting materials for preparing thecompounds of the invention are either commercially available or can beconveniently prepared by one of examples set forth below or otherwiseusing known chemistry procedures.

Examples 1-12,14-45, 47-50, 99-116

The compounds of Examples 1-50 and 99-116 were prepared following thebelow mentioned general procedure for Example 13 using compound 13 (a)or its analogue and reacting it with the followingbenzenesulfonylhydrazines: 4-Phenylbenzenesulfonyl hydrazine,4-t-butylbenzenesulfonyl hydrazine,4-methyl-3,4-dihydro-2i7-benzo[1,4]oxazine-7-sulfonyl hydrazine,5-(1-dimethylaminonaphthyl)sulfonyl hydrazine,2,4,6-trimethylbenzenesulfonyl hydrazine,3-chloro-6-methoxybenzenesulfonyl hydrazine,2,5-dimethoxybenzenesulfonyl hydrazine,4-(4-[1,2,3]thiadiazolyl)benzenesulfonyl hydrazine,3-bromobenzenesulfonyl hydrazine, 4-bromobenzenesulfonyl hydrazine,4-methylbenzenesulfonyl hydrazine, 4-methoxybenzenesulfonyl hydrazine,3-fluoro-4-chlorobenzenesulfonyl hydrazine,4-trifluoromethoxybenzenesulfonyl hydrazine, 4-fluorobenzenesulfonylhydrazine, 3-methoxybenzenesulfonyl hydrazine, 2-methylbenzenesulfonylhydrazine, 3-trifluoromethylbenzenesulfonyl hydrazine,2,4-dimethoxybenzenesulfonyl hydrazine,5-chloro-1,3-dimethyl-1H-pyrazolylsulfonyl hydrazine,3-methylbenzenesulfonyl hydrazine, 4-trifluoromethylbenzenesulfonylhydrazine, 2-trifluoromethylbenzenesulfonyl hydrazine,4-(pyrrolidin-1-sulfonyl)benzenesulfonyl hydrazine,2-chlorobenzenesulfonyl hydrazine, 5-(2-morpholin-4-yl)pyridylsulfonylhydrazine, 2-trifluoromethoxybenzenesulfonyl hydrazine,2,4-dichlorobenzenesulfonyl hydrazine, benzenesulfonyl hydrazine,3-difluoromethylbenzenesulfonyl hydrazine, 3-cyanobenzenesulfonylhydrazine, 4-cyanobenzenesulfonyl hydrazine,5-(2,3-dihydrobenzo[1,4]dioxinyl)sulfonyl hydrazine,2-(4-methylbenzenesulfonyl)-1-methyl hydrazine, 3-fluorobenzenesulfonylhydrazine, 3,4-difluorobenzenesulfonyl hydrazine,2,4-dimethylthiazol-5-ylsulfonyl hydrazine, 4-acetylbenzenesulfonylhydrazine, 2,6-difluorobenzenesulfonyl hydrazine,2-fluorobenzenesulfonyl hydrazine, 2,5-difluorobenzenesulfonylhydrazine, 1-(4-methylbenzenesulfonyl)-1-methyl hydrazine,2,6-dichlorobenzenesulfonyl hydrazine,2,6-ditrifluoromethylbenzenesulfonyl hydrazine,3,5-dimethylisoxazol-5-ylsulfonyl hydrazine, 4-nitrobenzenesulfonylhydrazine, (1-methylimidazol-4-yl)sulfonyl hydrazine, methylsulfonylhydrazine, 2-naphthylsulfonyl hydrazine, 4-isopropylbenzenesulfonylhydrazine, 2-phenylbenzenesulfonyl hydrazine,3,4-dichlorobenzenesulfonyl hydrazine, 3,5-dimethylbenzenesulfonylhydrazine, 1-naphthylsulfonyl hydrazine,2,5-bis(2,2,2-trifluoroethoxy)benzenesulfonyl hydrazine, and5-bromo-2-methoxybenzenesulfonyl hydrazine.

Example 13

Preparation ofN-2-(1,1,1,3,3,3-Hexafluoro-2-methyIpropyI)-2-[(4-difluoromethoxyphenyl)sulfonyl]hydrazine-1-carboxamide

a. Preparation of 1,1,1,3,3,3-Hexafluoro-2-isocyanato-2-methylpropane,compound 13(a)

A solution of trimethylsilylazide (26 mL, 180 mmol) was slowly addeddropwise to a solution of 2,2-bis(trifluoromethyl)propionyl fluoride (38g, 179 mmol) and benzyltriethylammonium chloride (0.065 g, 0.28 mmol) inxylenes (120 mL) at 0° C. Upon completion of the addition, the resultingmixture was heated at 110° C. After 4 h, the mixture was distilled at760 mm Hg, and the fraction boiling at 40-50° C. contained 13 (a). Yieldof the liquid product is 60%.

b. Preparation ofN-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-difluoromethoxyphenyl)sulfonyl]hydrazine-1-carboxamide

To a solution of 4-difluorobenzenesulfonyl chloride (60 mg, 0.25 mmol)in methylamine (25 mg, 0.25 mmol) in 1 mL of dry THF was added anhydroushydrazine (15 mg, 0.26 mmol) at room temperature. After stirring at roomtemperature for 2 h, a solution of1,1,1,3,3,3-hexafluoro-2-isocyanato-2-methylpropane (13a) (54 mg, 0.26mmol) in 1 mL of diethylether. The reaction mixture was stirred at roomtemperature for 12 h. The solvent was removed in vacuo, and the crudematerial subjected to reverse phase HPLC affording the product as awhite, waxy solid (83 mg, 75%).

Example 46

Preparation ofN-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-methylphenyl)sulfonyl]hydrazine-1-methylcarboxamide

To a solution ofN-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-methylphenyl)sulfonyl]hydrazine-1-carboxamide(100 mg, 0.254 mmol) prepared as described above, and cesium carbonate(165 mg, 0.51 mmol) in 1.6 mL of NMP was added iodomethane (17.5 yL,0.28 mmol). The yellow mixture was stirred at room temperature for 2 hbefore adding 5 mL of water. The mixture was extracted with EtOAc, andthe organic phase washed successively with water and brine. The organicphase was dried over MgSO4, and concentrated in vacuo. The crude productwas chromatographed on silica gel with 10% EtOAc in hexanes.

Example 51

Preparation of4-Phenylpiperazine-1-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-carboxamide.To 1-phenylpiperazine (0.04 mL, 0.25 mmol) was added1,1,1,3,3,3-hexafluoro-2-isocyanato-2-methylpropane (13a) (124 mg, 0 6mmol) in 1 mL of diethylether. The mixture was stirred at roomtemperature in a tightly capped vial for 12 h. The reaction mixture wassubjected to reverse phase HPLC (CH₃CN/H₂O) and the isolated productlyophilized to provide the product as a white solid.

Examples 52-98

The compounds of Examples 52-98 were prepared following the abovementioned general procedure for Example 51 using compound 13 (a) andreacting it with the following amines or anilines: morpholine,2-acetylaniline, piperidine, 3,4,5-trimethoxyaniline,4-trifluoromethylaniline, 4-methylpiperazine, 1-aminonaphthalene,2-chloroaniline, 4-phenylpiperidine, 2-phenylaniline,2,6-difluoroaniline, 2-amimobenzamide, 2-chloro-6-fluoroaniline,3-trifluoromethylaniline, 2-aminobenzenesulfonamide,5-amino(2,2,3,3-Tetrafluoro-2,3-dihydrobenzo[1,4]dioxane),3-trifluoromethoxyaniline, 4-trifluoromethoxyaniline,4-methylpiperidine, 2-aminonaphthalene, 2-fluoroaniline,2,6-dimethoxyaniline, 4-amino-3-trifluoromethoxybenzoic acid, aniline,3-cyanoaniline, 3-methoxyaniline, 2-(1,1,2,2-tetrafluoroethoxy)aniline,3-aminobenzenesulfonamide, 3-fluoroaniline, 4-bromoaniline,2-cyanoaniline, 4-cyanoaniline, 3-amino-2,2-difluorobenzo[1,3]dioxane,4-chloroaniline, 3-methylaniline, 4-aminobenzenesulfonamide,2,6-dibromoaniline, 2-methylaniline, 4-methylaniline, pyrrolidine,4-fluoroaniline, 2,4-dibromoaniline, azepane,4-bromo-2-trifluoromethoxyaniline, 2-trifluoromethoxyaniline,2-trifluoromethylaniline, and 2-methoxyaniline.

The synthesized compounds are summarized in Table A and Table A1.

TABLE A Example Number Structure Name 1

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-(phenyl)-phenylsulfonyl]hydrazine-1- carboxamide 2

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[(4-(2-methyl-2-propyl)-phenylsulfonyl]hydrazine-1-carboxamide 3

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[7-(4-methyl-3,4-dihydro-2H- benzo[1,4]oxazinyl)sulfonyl]hydrazine-1-carboxamide 4

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[5-(1-dimethylamino-naphthyl)sulfonyl]hydrazine-1-carboxamide 5

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2,4,6-trimethylphenyl)sulfonyl]hydrazine- 1-carboxamide 6

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[(3-chloro-6-methoxyphenyl)sulfonyl]hydrazine-1- carboxamide 7

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3,6-dimethoxyphenyl)sulfonyl]hydrazine- 1-carboxamide 8

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[(4-(4-[1,2,3]thiadiazolyl)phenyl)sulfonyl]hydrazine- 1-carboxamide 9

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3-bromophenyl)sulfonyl]hydrazine-1- carboxamide 10

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-bromophenyl)sulfonyl]hydrazine-1- carboxamide 11

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-methylphenyl)sulfonyl]hydrazine-1- carboxamide 12

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-methoxyphenyl)sulfonyl]hydrazine-1- carboxamide 13

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[(4-difluoromethoxyphenyl)sulfonyl]hydrazine-1- carboxamide 14

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[(3-fluoro-4-chloro-phenyl)sulfonyl]hydrazine-1-carboxamide 15

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[(4-trifluoromethoxyphenyl)sulfonyl]hydrazine-1- carboxamide 16

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-fluoro-phenyl)sulfonyl]hydrazine-1 carboxamide 17

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3-methoxyphenyl)sulfonyl]hydrazine-1- carboxamide 18

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2-methylphenyl)sulfonyl]hydrazine-1- carboxamide 19

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[(3-trifluoromethylphenyl)sulfonyl]hydrazine-1- carboxamide 20

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2,4-dimethoxyphenyl)sulfonyl]hydrazine- 1-carboxamide 21

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[2-(5-chloro-1,3-dimethyl-1H-pyrazolyl)sulfonyl]hydrazine-1-carboxamide 22

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3-methylphenyl)sulfonyl]hydrazine-1- carboxamide 23

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[(4-trifluoromethylphenyl)sulfonyl]hydrazine-1- carboxamide 24

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[(2-trifluoromethylphenyl)sulfonyl]hydrazine-1- carboxamide 25

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[(4-(pyrrolidin-1-sulfonyl)phenylsulfonyl]hydrazine-1- carboxamide 26

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2-chlorophenyl)sulfonyl]hydrazine-1- carboxamide 27

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[2-(5-morpholin-4-yl)pyridylsulfonyl]hydrazine-1-carboxamide 28

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[(2-trifluoromethoxyphenyl)sulfonyl]hydrazine-1- carboxamide 29

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2,4-dichlorophenyl)sulfonyl]hydrazine-1- carboxamide 30

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[phenylsulfonyl]hydrazine-1-carboxamide 31

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[(3-difluoromethoxyphenyl)sulfonyl]hydrazine-1 carboxamide 32

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3-cyanophenyl)sulfonyl]hydrazine-1 carboxamide 33

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-cyanophenyl)sulfonyl]hydrazine-1- carboxamide 34

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[5-(2,3-dihydrobenzo[1,4]dioxinyl)sulfonyl]hydrazine- 1-carboxamide 35

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-methylphenyl)sulfonyl]-1- methylhydrazine-1-carboxamide 36

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3-fluorophenyl)sulfonyl]hydrazine-1- carboxamide 37

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(3,4-difluorophenyl)sulfonyl]hydrazine-1- carboxamide 38

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[(2,4-dimethylthiazol-5-yl)sulfonyl]hydrazine-1-carboxamide 39

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-acetylphenyl)sulfonyl]hydrazine-1- carboxamide 40

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2,6-difluorophenyl)sulfonyl]hydrazine-1- carboxamide 41

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2-fluorophenyl)sulfonyl]hydrazine-1- carboxamide 42

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2,5-difluorophenyl)sulfonyl]hydrazine-1- carboxamide 43

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-methylphenyl)sulfonyl]-2- methylhydrazine-1-carboxamide 44

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(2,6-dichlorophenyl)sulfonyl]hydrazine-1- carboxamide 45

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[(2,6-ditrifluoromethylphenyl)sulfonyl]hydrazine-1- carboxamide 46

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-methylphenyl)sulfonyl]hydrazine-1- methylcarboxamide 47

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)- 2-[(3,5-dimethylisoxazol-5-yl)sulfonyl]hydrazine-1-carboxamide 48

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(4-nitrophenyl)sulfonyl]hydrazine-1- carboxamide 49

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[(1-methylimidazol-4-yl)sulfonyl]hydrazine- 1-carboxamide 50

N-2-(1,1,1,3,3,3-Hexafluoro-2-methylpropyl)-2-[methylsulfonyl]hydrazine-1-carboxamide 51

4-Phenylpiperazine-1-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-carboxamide 52

4-Morpholino-1-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-carboxamide 53

1-(2-Acetylphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 54

1-Piperidino-1-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-carboxamide 55

1-(2,2,2-trifluoro-1-methyl-1- trifluoromethylethyl)-3-(3,4,5-trimethoxyphenyl)-urea 56

1-(4-Trifluoromethylphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 57

4-Methylpiperazine-1-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-carboxamide 58

1-Naphthalen-1-yl-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 59

1-(4-Chlorophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 60

4-Phenylpiperidin-1-yl-1-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-carboxamide 61

1-(2-Phenyl(phenyl))-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 62

1-(2,6-Difluorophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 63

2-[3-(1,1-Bis-trifluoromethylethyl)- ureido]benzamide 64

1-(2-Chloro-6-fluorophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 65

1-(3-Trifluoromethylphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 66

2-[3-(1,1-Bis-trifluoromethylethyl)- ureido]benzenesulfonamide 67

1-(2,2,3,3-Tetrafluoro-2,3- dihydrobenzo[1,4]dioxin-5-yl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 68

1-(3-Trifluoromethoxyphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 69

1-(4-Trifluoromethoxyphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 70

4-Methyl-1-piperidine-1-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-carboxamide 71

1-Naphthalen-2-yl-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 72

1-(2-fluorophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 73

1-(2,6-Dimethoxyphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 74

3-Trifluormethoxy-4-[3-(1,1-bis- trifluoromethylethyl)-ureido]benzoicacid 75

1-Phenyl-3-(2,2,2-trifluoro-1-methyl-1- trifluoromethylethyl)-urea 76

1-(3-Cyanophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 77

1-(3-Methoxyphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 78

1-(2-(1,1,2,2-Tetrafluoroethoxy)phenyl)-3- (2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 79

3-[3-(1,1-Bis-trifluoromethylethyl)- ureido]benzenesulfonamide 80

1-(3-fluorophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 81

1-(4-Bromophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 82

1-(2-Cyanophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 83

1-(4-Cyanophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 84

1-(2,2-Difluorobenzo[1,3]dioxol-4-yl)-3- (2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 85

1-(4-Chlorophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 86

1-(3-Methylphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 87

4-[3-(1,1-Bis-trifluoromethylethyl)- ureido]benzenesulfonamide 88

1-(2,6-Dibromophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 89

1-(2-Methylphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 90

1-(4-Methylphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 91

1-Pyrrolidinyl-1-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-carboxamide 92

1-(4-Fluorophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 93

1-(2,4-Dibromophenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 94

Azepane-1-carboxylic acid (2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-amide 95

1-(4-Bromo-2-trifluoromethoxyphenyl)-3- (2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 96

1-(2-Trifluoromethoxyphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 97

1-(2-Trifluoromethylphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea 98

1-(2-Methoxyphenyl)-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)-urea

TABLE A1 Com- Molecular pound Molecular Structure Weight Analytical DataChemical Name 99

435.39 ¹H NMR in DMSO-d6: δ (ppm) 9.48 (s, 1H), 8.32 (s, 1H), 7.00 (s,2H), 6.81 (s, 1H), 2.56 (s, 6H), 2.24 (m 5H), 0.74 (t, 3H)1-[1,1-bis(trifluoromethyl)propyl]- 3-[(2,4,6-trimethylphenyl)sulfonylamino] urea 100

449.41 ¹H NMR in DMSO-d6: δ (ppm) 9.61 (s, 1H), 8.35 (s, 1H), 7.72 (d,2H), 7.60 (d, 2H), 6.85 (s, 1H), 2.36 (q, 2H), 1.30 (s, 9H), 0.80 (t,3H) 1-[1,1-bis(trifluoromethyl)propyl]- 3-[(4-tert-butylphenyl)sulfonylamino]urea 101

453.36 ¹H NMR in DMSO-d6: δ (ppm) 9.34 (s, 1H), 8.30 (s, 1H), 7.15- 7.23(m, 3H), 6.81 (s, 1H), 3.85 (s, 3H), 3.74 (s, 3H), 2.28 (q, 2H), 0.84(t, 3H) 1-[1,1-bis(trifluoromethyl)propyl]- 3-[(2,5-dimethoxyphenyl)sulfonylamino] urea 102

443.37 ¹H NMR in CD₃OD: δ (ppm) 8.24 (s, 1H), 8.03-8.05 (m, 2H), 7.97(d, 1H), 7.87 (d, 1H), 7.63- 7.70 (m, 2H), 2.24 (d, 2H), 0.73 (s, 3H);Mass Spec: 444.3 (M + H)⁺ 1-[1,1-bis(trifluoromethyl)propyl]-3-(2-naphthylsulfonylamino)urea 103

435.39 ¹H NMR in DMSO-d6: δ (ppm) 9.62 (s, 1H), 8.34 (s, 1H), 7.71 (d,2H), 7.45 (d, 2H), 6.85 (s, 1H), 2.97 (septet, 1H), 2.24 (q, 2H), 1.21(d, 6H), 0.80 (t, 3H) 1-[1,1-bis(trifluoromethyl)propyl]- 3-[(4-isopropylphenyl)sulfonylamino] urea 104

457.78 ¹H NMR in DMSO-d6: δ (ppm) 9.57 (s, 1H), 8.38 (s, 1H), 7.69 (dd,1H), 7.64 (d, 1H), 7.26 (d, 1H), 6.85 (s, 1H), 3.91 (s, 3H), 2.25 (q,2H), 0.82 (t, 3H) 1-[1,1-bis(trifluoromethyl)propyl]-3-[(5-chloro-2-methoxy- phenyl)sulfonylamino]urea 105

469.40 ¹H NMR in DMSO-d6: δ (ppm) 9.34 (s, 1H), 8.36 (s, 1H), 7.98 (d,1H), 7.67 (t, 1H), 7.55 (t, 1H), 7.48-7.49 (m, 2H), 7.36- 7.38 (m, 3H),7.31 (d, 1H), 6.93 (s, 1H), 2.26 (q, 2H), 0.82 (t, 3H)1-[1,1-bis(trifluoromethyl)propyl]- 3-[(2-phenylphenyl)sulfonylamino]urea 106

462.20 ¹H NMR in DMSO-d6: δ (ppm) 10.04 (s, 1H), 8.49 (s, 1H), 7.91 (d,1H), 7.87 (d, 1H), 7.75 (dd, 1H), 7.03 (s, 1H), 2.21 (q, 2H), 0.81 (t,3H) 1-[1,1-bis(trifluoromethyl)propyl]- 3-[(3,4-dichlorophenyl)sulfonylamino]urea 107

472.20 ¹H NMR in DMSO-d6: δ (ppm) 9.86 (s, 1H), 8.39 (s, 1H), 7.80 (d2H) 7.70 (d 2H) 6.93 (s 1H), 2.23 (q, 2H), 0.81 (t, 3H)1-[1,1-bis(trifluoromethyl)propyl]- 3-[(4-bromophenyl)sulfonylamino]urea 108

459.31 ¹H NMR in DMSO-d6: δ (ppm) 9.77 (s, 1H), 8.39 (s, 1H), 7.84 (d,2H), 7.39 (t, 1H), 7.36 (d, 2H), 6.90 (s, 1H), 2.24 (q, 2H), 0.81 (t,3H) 1-[1,1-bis(trifluoromethyl)propyl]- 3-[[4-(difluoromethoxy)phenyl]sulfonyl- amino]urea 109

421.36 ¹H NMR in DMSO-d6: δ (ppm) 9.64 (s, 1H), 8.35 (s, 1H), 7.40 (s,2H), 7.30 (s, 1H), 6.85 (s, 1H), 2.33 (s, 6H), 2.26 (q, 2H), 0.84 (t,3H) 1-[1,1-bis(trifluoromethyl)propyl]- 3-[(3,5-dimethylphenyl)sulfonylamino] urea 110

486.44 ¹H NMR in DMSO-d6: δ (ppm) 9.96 (s, 1H), 8.50 (d, 1H), 8.40 (s,1H), 8.30 (d, 1H), 8.16 (d, 1H), 7.61 (q, 2H), 7.26 (d, 1H), 6.67 (s,1H), 2.83 (s, 6H), 2.12 (q, 2H), 0.71 (t, 3H)1-[1,1-bis(trifluoromethyl)propyl]- 3-[[5-(dimethylamino)-1-naphthyl]sulfonylamino]urea 111

443.37 ¹H NMR in DMSO-d6: δ (ppm) 10.00 (s, 1H), 8.66 (d, 1H), 8.40 (s,1H), 8.26 (d, 1H), 8.17 (d, 1H), 8.09 (d, 2H), 7.61-7.74 (m 3H), 6.72(s, 1H), 2.11 (q, 2H), 0.70 (t, 3H) 1-[1,1-bis(trifluoromethyl)propyl]-3-(1-naphthylsulfonylamino)urea 112

464.39 ¹H NMR in DMSO-d6: δ (ppm) 9.40 (s, 1H), 8.25 (s, 1H), 7.02 (d,1H), 7.00 (s, 1H), 6.81 (d, 1H), 4.29 (m, 2H), 3.31 (m, 2H), 2.87 (s,3H), 2.29 (q, 2H), 0.86 (t, 3H) 1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-methyl-2,3-dihydro-1,4- benzoxazin-7-yl)sulfonylamino] urea 113

589.35 ¹H NMR in DMSO-d6: δ (ppm) 9.35 (s, 1H), 8.47 (s, 1H), 7.37 (m,3H), 6.90 (s, 1H), 4.72-4.90 (m, 4H), 2.23 (q, 2H), 0.77 (t, 3H)1-[[2,5-bis(2,2,2- trifluoroethoxy)phenyl]sulfonyl-amino]-3-[1,1-bis(trifluoromethyl) propyl]urea 114

477.41 ¹H NMR in DMSO-d6: δ (ppm) 9.86 (s, 1H), 9.82 (s, 1H), 8.43 (s,1H), 8.36 (d, 2H), 7.95 (d, 2H), 6.93 (s, 1H), 2.22 (q, 2H), 0.79 (t,3H) 1-[1,1-bis(trifluoromethyl)propyl]- 3-[[4-(thiadiazol-4-yl)phenyl]sulfonylamino]urea 115

502.23 ¹H NMR in DMSO-d6: δ (ppm) 9.57 (s, 1H), 8.41 (s, 1H), 7.80 (dd,1H), 7.75 (d, 1H), 7.21 (d, 1H), 6.84 (s, 1H), 3.91 (s, 3H), 2.25 (q,2H), 0.82 (t, 3H) 1-[1,1-bis(trifluoromethyl)propyl]-3-[(5-bromo-2-methoxy- phenyl)sulfonylamino]urea 116

469.40 ¹H NMR in CD₃OD: δ (ppm) 7.94-7.97 (m, 2H), 7.81 (d, 2H),7.65-7.68 (m, 2H), 7.46-7.50 (m, 2H), 7.41-7.43 (m 1H), 2.34 (d, 2H),0.89 (t, 3H) 1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-phenylphenyl)sulfonylamino] urea

Assay 1

Approximately 400,000 compounds from the compound library were tested inthis assay. Assay plates were set up as follows. Vero cells were platedat 80% confluency on 96-well plates. Test compounds (80 per plate) fromthe library were added to wells at a final concentration of 5 uM.Tacaribe virus (TRVL 11573) was then added at a virus dilution thatwould result in 90% CPE after 5 days (pre-determined as an 800-folddilution of the virus stock; multiplicity of infection[MOT]approximately 0.001). Plates were incubated at 37° C. and 5% CO2for 5 days, then fixed with 5% glutaraldehyde and stained with 0.1%crystal violet. The extent of virus CPE was quantified spectrometricallyat OD570 using a Molecular Devices VersaMax Tunable Microplate Reader.The inhibitory activity of each compound was calculated by subtractingfrom the OD570 of test compound well from the average OD570 ofvirus-infected cell wells, then dividing by the average OD570 ofmock-infected cell wells. The result represents the percent protectionagainst Tacaribe virus CPE activity conferred by the compound.“Hits” inthis assay were defined as compounds that inhibited virus-induced CPE bygreater than 50% at the test concentration (5 (J.M). Of theapproximately 400,000 compounds screened in the Tacaribe virus HTScampaign, 2,347 hits were identified (0.58% hit rate).

Quality hits are defined as inhibitor compounds (hits) that exhibitacceptable chemical structures, antiviral potency and selectivity, andspectrum of antiviral activity. Specifically, compounds identified ashits in HTS assays (described above) were evaluated against fourcriteria: (i) chemical tractability, (ii) inhibitory potency, (iii)inhibitory selectivity and, (iv) antiviral specificity. Based on the HTSparameters, all hits have EC50 values <5 uM. The chemical structures ofcompounds that met this initial criterion were visually examined forchemical tractability. A chemically tractable compound is defined as anentity that is synthetically accessible using reasonable chemicalmethodology, and which possesses chemically stable functionalities and(potential) drug-like qualities. Hits that passed this medicinalchemistry filter were evaluated for their inhibitory potency. EC50values were determined from a plot of the compound inhibitory activitytypically across eight compound concentrations (50, 15, 5, 1.5, 0.5,0.15, 0.05 and 0.015 uM). To assess whether the hit is a selectiveinhibitor, the effect on cellular functions was determined using astandard cell proliferation assay. A 50% cytotoxicity concentration(CC50) was determined using a tetrazolium-based colorimetric method,which measures the in situ reduction of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) toinsoluble blue formazan crystals by mitochondrial enzymes inmetabolically active cells. Solubilized crystals were quantifiedspectrometrically. Using the EC50 and CC50 values, a Selective Index(SI) was calculated (SI=CC50/EC50). Hits with SI values of at least 10were considered further.

The specificity of the antiviral activity exhibited by hit compounds wasdetermined by testing the compounds against a number of related andunrelated viruses. Compounds are tested against a variety of unrelatedDNA (HSV, CMV, vaccinia virus) and RNA (RSV, rotavirus, Rift Valleyfever, Ebola virus, Ebola GP-pseudotype, Lassa GP-pseudotype, HIVenv-pseudotype) viruses. Compounds that will be selected for furtherdevelopment are thosejhâ are selective against the selected originaltarget virus and inactive against unrelated viruses.

The results are summarized in Table B and Table B1.

TABLE B Tacaribe EC₅₀ Candide I A = <0.5 μM A = <0.5 μM B = 0.5 to <1.0μM B = 0.5 to <1.0 μM Example C = 1.0 to <5 μM C = 1.0 to <5 μM Number D= ≧5 μM D = ≧5 μM 1 A 2 A 3 A 4 A 5 A 6 A 7 A 8 A 9 A 10 A 11 A 12 A 13A 14 B 15 B 16 B 17 B 18 B 19 B 20 B 21 B C 22 B 23 B 24 C 25 C 26 C 27C 28 C 29 C 30 C 31 C 32 C 33 C 34 C 35 C 36 C 37 C 38 C 39 C D 40 C 41C 42 C 43 C 44 C 45 C 46 D 47 D 48 D 49 D 50 D

TABLE B1 Tacaribe EC₅₀ A = <0.5 μM B = 0.5 to <1.0 μM Example C = 1.0 to<5 μM Number D = ≧5 μM 51 A 52 A 53 B 54 B 55 B 56 B 57 C 58 C 59 C 60 C61 C 62 C 63 C 64 C 65 C 66 C 67 C 68 C 69 C 70 C 71 C 72 C 73 C 74 C 75C 76 C 77 C 78 C 79 C 80 C 81 C 82 C 83 C 84 C 85 D 86 D 87 D 88 D 89 D90 D 91 D 92 D 93 D 94 D 95 D 96 D 97 D 98 D

Assay 2

A chemical library was created and screened that represents a broad andwell-balanced collection of 400,000 compounds accumulated over a numberof years from a variety of distinct sources. The library achieves broadcoverage across property space involving the following chemicaldescriptors: calculated logarithm of n-octanol/water partitioncoefficient (ClogP), polar (water-accessible surface area (PSA),globularity (three dimensional structure) and molecular weight (average:394.5 daltons).

Cells and Viruses

Vero (African green monkey kidney epithelial, ATCC #CCL-81) cells weregrown in Eagle's minimum essential medium (MEM, Gibco) supplemented with2 mM L-glutamine, 25 μg/ml gentamicin, and 10% heat-inactivated fetalbovine serum (FBS). For infection medium (IM), the serum concentrationwas reduced to 2%. HEp-2 cells (human carcinoma of the larynxepithelial; ATCC #CCL-23) were cultured in MEM containing 10%heat-inactivated FBS and 1% penicillin/streptomycin. MRC-5 cells (humannormal lung fibroblast; ATCC #CCL-171) were cultured in MEM containing10% heat-inactivated FBS, 1% penicillin/streptomycin, 1% L-glutamine(Invitrogen 25030-081), 1% Non-Essential Amino Acids (Invitrogen#11140-050), 1% sodium pyruvate (Invitrogen #11360-070), and 2% sodiumbicarbonate. MA104 cells (epithelial African green monkey kidney, ATCCCRL-2378.1) were cultured in MEM with 1% penicillin/streptomycin, 1%L-glutamine, 1% Non-Essential Amino Acids, 1% sodium pyruvate, and 2%sodium bicarbonate and 62.5 ug/ml trypsin and no serum during virusinfection. All cell lines were incubated at 37° C. and 5% CO₂.Respiratory syncytial virus (RSV; A isolate), lymphocyticchoriomeningitis virus (LCMV; Armstrong E350 isolate), cytomegalovirus(CMV; AD-169 isolate), herpes simplex virus 1 (HSV-1; KOS isolate),Vaccinia virus (Strain WR), Tacaribe virus (strain TRVL 11573) androtavirus (strain WA) were obtained from ATCC (#VR-1422, #VR-1540,#VR-134, #VR-538, #VR-1493, #VR-1354, #VR-114, and # VR-2018respectively). Candid 1 and Amapari BeAn 70563 were obtained from Dr.Robert Tesh at the University of Texas Medical Branch (Galveston, Tex.).Work done with BSL 4 viruses (Lassa, Machupo, Guanarito, and Junin) aswell as severe acute respiratory syndrome-associated coronavirus(SARS-CoV) was conducted by collaborators at USAMRIID (Fort Detrick,Md.).

Antiviral Assays for Specificity Screening: Cytopathic Effect (“CPE”)Assay, Virus Plaque Reduction Assay, and ELISA

A viral CPE assay was used to evaluate the antiviral effect of compoundsagainst Tacaribe virus (Vero cells), Candid-1 vaccine virus (Verocells), Amapari virus (Vero cells), SARS-CoV (Vero cells), HSV-1 (Verocells), RSV (HEp-2 cells), vaccinia virus (Vero cells), and Rotavirus(MA104). An enzyme-linked immunosorbent assay (“ELISA”) was used toevaluate the antiviral effect of compounds against CMV (MRC-5 cells) andLCMV (Vero cells). All of these assays were carried out in theappropriate media containing 2% heat-inactivated FBS. Ninety-six-wellcell culture plates were seeded 24 hours before use with 1.5×10⁴ (Vero),2.2×10⁴ (HEp-2 and MA104), and 4.5×10⁴ (MRC-5) cells per well. Forcompound susceptibility testing, compounds (solubilized with 100% DMSO)were added to duplicate wells at final concentrations of 50, 15.8, 5,1.6, 0.5, 0.16, 0.05, 0.016 and 0 μM.

The final concentration of DMSO in the assays was 0.5%. Virus stockswere titrated in a separate experiment to determine the concentrationthat resulted in 90% destruction of the cell monolayer (CPE assay) after3 days (HSV-1, Rotavirus and vaccinia) or 4 days (SARS-CoV, RSV,Tacaribe virus, Candid 1 vaccine virus and Amapari virus) or theconcentration that generated an ELISA signal of 2.5 at an opticaldensity of 650 nm (OD₆₅₀) after 3 days (LCMV) or 4 days (CMV). Thesepre-established dilutions of virus were added to wells containing serialdilutions of compound. Uninfected cells and cells receiving viruswithout compound were included on each assay plate. In addition,reference agents, when available, were included on each assay plate(gancyclovir for HSV-1 and CMV, Sigma #G2536; ribavirin for LCMV andRSV, Sigma #R9644; and rifampicin for vaccinia virus, Sigma #R3501).Plates were incubated at 37′C and 5% CO₂ for either 3 days (HSV-1,Rotavirus, LCMV, Vaccinia virus) or 4 days (Tacaribe virus, Amaparivirus, Candid 1 virus, SARS-CoV, RSV, and CMV). HSV-1, SARS-CoV,Rotavirus, Vaccinia virus, RSV, Tacaribe virus, Amapari virus, Candid 1vaccine virus infected plates were processed for crystal violet stainingwhile plates infected with CMV and LCMV were processed for ELISAanalysis.

For crystal violet staining, the plates were fixed with 5%glutaraldehyde and stained with 0.1% crystal violet. After rinsing anddrying, the optical density at 570 nm (OD₅₇₀) was measured using aMicroplate Reader. For ELISA analysis, the medium from the LCMV andCMV-infected plates was removed and the cells were fixed with 100%methanol (Fisher, CAS #67-56-1, HPLC grade) for 20 minutes at roomtemperature. The methanol solution was removed and the plates werewashed 3 times with PBS. Non-specific binding sites were blocked by theaddition of 130 μL of Superblock Blocking Buffer (Pierce #37515) for 1hour at 37° C. The blocking agent was removed and the wells were washed3 times with PBS. Thirty μL of a 1:20 dilution of LCMV Nuclear Protein(NP) specific monoclonal antibody (generous gift of Juan Carlos de laTorre, The Scripps Research Institute, La Jolla Calif.) or 30 μL of a1:200 dilution of CMV (protein 52 and unique long gene 44 product)specific cocktail monoclonal antibodies (Dako, #M0854) in SuperblockBlocking Buffer containing 0.1% Tween-20 was added.

Following 1 hour incubation at 37° C., the primary antibody solution wasremoved and the wells were washed 3 times with PBS containing 0.1%Tween-20. Forty μL of goat anti-mouse horseradish peroxidase conjugatedmonoclonal antibody (Bio-Rad #172-1011) diluted 1:4000 (LCMV) or 1:400(CMV) in Superblock Blocking Buffer containing 0.1% Tween-20 was addedto the wells and the plates were incubated for 1 hour at 37° C. Thesecondary antibody solution was removed and the wells were washed 5times with PBS. The assay was developed for 15 minutes by the additionof 130 μL of 3,3′,5,5-tetramethylbenzidine substrate (Sigma #T0440) toquantify peroxidase activity. The OD₆₅₀ of the resulting reactionproduct was measured using a Molecular Devices Kinetic Microplate Readerwith a 650 nm filter.

Antiviral activity against Tacaribe virus was evaluated by threemethods: CPE Assay, Plaque Reduction, and Virus Yield Inhibition Assay.For the HTS CPE Assay, Vero cells were plated at 80% confluency on96-well plates. Test compounds (80 per plate) from the library wereadded to wells at a final concentration of 5 μM. Tacaribe virus was thenadded at a virus dilution that would result in 90% CPE after 5 days(multiplicity of infection (“MOI”) approximately 0.001). Plates wereincubated at 37° C. and 5% CO₂ for 5 days, then fixed with 5%glutaraldehyde and stained with 0.1% crystal violet. The extent of virusCPE was quantified spectrometrically at OD₅₇₀ using an EnvisionMicroplate Reader. The inhibitory activity of each compound wascalculated by subtracting from the OD₅₇₀ of test compound well from theaverage OD₅₇₀ of virus-infected cell wells, then dividing by the averageOD₅₇₀ of mock-infected cell wells. The result represents the percentprotection against Tacaribe virus CPE activity conferred by eachcompound. “Hits” in this assay were defined as compound that inhibitedvirus-induced CPE by greater than 50% at the test concentration (5 μM).Hits that possessed chemical tractability were further evaluated fortheir inhibitory potency. The inhibitory concentration 50% (EC₅₀) valueswere determined from a plot of the compound inhibitory activityfollowing the CPE assay across eight compound concentrations (50, 15, 5,1.5, 0.5, 0.15, 0.05 and 0.015 μM). All determinations were performed induplicate.

In the Plaque Reduction Assay, Vero cell monolayers grown in 6-wellplates were infected with about 50 PFU/well in the absence or presenceof various concentrations of the compounds. After 1 h of virusadsorption at 37° C., residual inoculum was replaced by a 50:50 mix of1% seaplaque agarose (in de-ionized water) and 2× MEM. Plaques werecounted after 5-7 days of incubation at 37° C. The EC₅₀ was calculatedas the compound concentration required to reduce virus plaque numbers by50%. Under BSL 4 conditions at USAMRIID the plaque reduction assays(with Lassa, Machupo, Guanarito, and Junin viruses) were performed asfollows: 200 PFU of each virus was used to infect Vero cells. Aftervirus adsorption, cell monolayers were rinsed and overlaid with completemedium containing 1% agarose and either lacking test compound or withdifferent concentrations ranging from 15 μM to 0.05 μM. After 5 daysincubation at 37° C., the monolayers were stained with neutral red andthe numbers of plaques were counted.

In Virus Yield Reduction Assays, Vero cells grown in 24-well plates wereinfected with Tacaribe virus at a multiplicity of infection (“MOI”) of0.1 in the presence of different concentrations of the compounds, twowells per concentration. After 48 h of incubation at 37° C. virus washarvested and the virus yields were determined by plaque formation inVero cells. The EC₅₀ values were calculated as indicated above andsimilar calculations were performed to determine EC90 and EC99.

Cytotoxicity Assay

Cell viability was measured by a cell proliferation assay to determine acompound's effect on cellular functions so that a 50% cytotoxicityconcentration (CC₅₀) could be calculated; the ratio of this value to theEC₅₀ is referred to as the selective index (S.I.=CC₅₀/EC₅₀). Two typesof assays were used to determine cytotoxicity. One was a colorimetricmethod that measures the reduction of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), andthe other uses fluorimetry to measure the reduction of resazurin (AlamarBlue). Both methods produced similar data. Confluent cultures in 96-wellplates were exposed to different concentrations of the compounds, withtwo wells for each concentration, using incubation conditions equivalentto those used in the antiviral assays.

Medicinal Chemistry

Several potent compounds were identified by the Tacaribe HTS and weregrouped into several clusters of structure type. One cluster ofcompounds, with ST-336 (FW=407.3) representing the prototype based onantiviral activity and chemical tractability, was chosen for furtherdevelopment. Through retrosynthetic analysis of ST-336, it wasdetermined that a library of analogues could be prepared convergently ina single synthetic step by combining an isocyanate with an acylhydrazide. Using this chemistry, 165 analogues were prepared and themost potent examined for in vitro metabolism (S9).

Time of Addition Experiment

This experiment was designed to characterize the mechanism of action ofthe anti-viral compounds. Vero cells were grown in 24 well cultureplates. The medium was removed when the cells reached 70-80% confluencyand replaced with infection medium. Cells were infected with Tacaribevirus at MOI=0.1. After 1 hour adsorption, the viral inoculum wasremoved and replaced with fresh infection medium. Duplicate wells weretreated with 3 μM ST-336 1 h prior to infection, at the time ofinfection or at specific times post infection (from 1 to 20 h p.i.).Control infected cell cultures were treated with drug vehicle (DMSO)only. ST-336 was removed 1 hour post absorption and the monolayer waswashed twice with cold PBS-M and replaced with fresh infection medium.The cells were harvested at 24 h p.i. and were titrated as describedabove.

In a separated experiment, Vero cells plated in a 6 well dish wereinfected with Tacaribe virus at MOI=4. Absorption was carried out for 1hour. Three μM of ST-336 was added for 1 hour at 1 hour beforeinfection, during infection, and 1 hour following infection. Followingdrug addition and virus infection, monolayers were washed 3 times withcomplete media. Four hours following last drug addition, monolayers wereoverlaid with 1% agarose without compound until plaques developed. At 5to 7 days post infection, monolayers were fixed, crystal violet stainedand plaque numbers counted.

Assay for Compound Binding to Intact Virus

This experiment was designed to test the binding/fusion inhibitoryproperties of ST-336 towards Tacaribe virus. Vero cells were grown inMEM with 2% fetal calf serum. For this experiment, cells were grown to70-80% confluency in 24-well culture plates. In one set of tubesTacaribe virus (4000 pfu) was treated with 1% DMSO, serially dilutedtenfold in infection medium and treated with the specific concentrationsof ST-336 (400 pfu+0.5 μM ST-336, 40 pfu+0.05 μM ST-336) or DMSO only(400 pfu or 40 pfu+DMSO). In another set of tubes Tacaribe virus (4000pfu) was treated with 5 μM ST-336 then serially diluted tenfold ininfection medium. The suspensions were plated in wells and afteradsorption for one hour inocula were removed and overlaid with 0.5%Seaplaque agarose in MEM. The plate was incubated at 37° C. untilcytopathic effect was observed in the DMSO control well. The cells werefixed with 5% gluteraldehyde and stained with 0.1% crystal violet forplaque visualization.

Another assay employed to test the binding properties of ST-336 topre-fusion F-proteins on virions was a dialysis experiment. PurifiedTacaribe virus (1000 pfu) was incubated with 5 μM of ST-336 or 0.5%DMSO. The suspensions were dialyzed overnight at 4° C. in a dialysischamber. Twenty four hours post dialysis viral suspensions were titratedon Vero cells. Post one hour adsorption, inocula were removed and a 0.5%Seaplaque agarose in MEM overlay was applied. The plate was incubated at37° C. until cytopathic effect was observed. The cells were fixed with5% gluteraldehyde and stained with 0.1% crystal violet. To confirmabsence of free drug in dialysed virus-drug sample, virus was spiked indialysed mixture at time of infection and plaques developed as describedabove.

Isolation of Drug Resistant Variant Viruses

Initially, single plaques of WT Tacaribe virus was isolated. For thisplaque-purification Vero cells in a 6-well plate were infected with 50pfu/well of WT Tacaribe virus for 1 hour at 37° C. Following virusadsorption the inoculum was removed and each well was overlaid with 0.5%Seaplaque agarose in MEM and incubated at 37° C. until plaques werevisible (5-7 days). Four plaques were picked and further amplified inVero cells in a 24-well plate until CPE developed (5-7 days).Virus-infected cell extracts were harvested by scraping cells into themedia and then collected in 1.5-ml microcentrifuge tubes. Eachplaque-purified isolate was further amplified in 150 mm plates, and theneach virus stock that originated from one virus plaque was titrated.

For the isolation of compound-resistant Tacaribe virus variants, eachwild type plaque-purified isolate was titrated in the presence of 3 μMST-336 as described. Vero cells in a 6-well plate were infected with10⁴-10⁶ pfu/well in media containing 3 μM ST-336 for 1 hour, then thecells were overlaid with 0.5% seaplaque agarose in MEM containing 3 μMST-336 and incubated until plaques formed. Plaques were picked and usedto infect Vero cells in a 24-well plate without compound. When CPEdeveloped the infected wells were harvested. Each drug-resistant isolatewas then titrated on a 96-well plate in 0.5 log dilutions, starting with25 μL of pure virus stock, without compound and with 1 μM and 3 μMST-336. Each mutant went through several rounds of plaque purificationbefore final virus stocks were made.

Sequencing

RNA was extracted from each of the Tacaribe WT isolates (1-4) and fourof the drug resistant isolates (DR#1-4) and used for reversetranscription PCR. Primers specific to the GPC (Tac-forward: 5′GCCTAACTGAACCAGGTGAATC (SEQ ID NO:1) and Tac-reverse: 5′TAAGACTTCCGCACCACAGG (SEQ ID NO:2)) from Tacaribe were used foramplification and sequencing.

Solubility

Two tests were used to assess compound solubility: solubility in cellculture medium with and without various concentrations of serum andsolubility in aqueous buffer at pH 7.4. The solutions were stirredovernight and then filtered through an Amicon Centrifree YM-30 columnwith a 30,000 MW cut off to remove potentially precipitated compound andcompound bound to protein. The compound was quantified by LC/MS or UVspectrometry.

Stability

In vitro metabolic stability was determined by Absorption Systems(Exton, Pa.) using the 9000×g supernatant (S9) of homogenized liver fromvarious species as a source of oxidative conjugation enzymes (e.g.,cytochromes P450, UdP-glucuronosyl transferase) that are known to be theprimary pathways of biotransformation for most drugs. The metabolicstability was measured as the persistence of parent compound overincubation time in the S9 fractions by mass spectrometry. Briefly,human, rat, mouse and guinea pig S9 fractions were obtained fromXenotech (Lenexa, Kans.). The reaction mixture, minus cofactorcocktails, was prepared (1 mg/ml liver S9 fractions, 1 mM NADPH, 1 mMUDPGA, 1 mM PAPS, 1 mM GSH, 100 mM potassium phosphate pH 7.4, 10 mMmagnesium chloride, 10 μM test article) and equilibrated at 37° C. for 3min. An aliquot of reaction mixture was taken as a negative control. Thereaction was initiated by the addition of cofactor cocktails to thereaction mixture only, and then the reaction mixture and negativecontrol were incubated in a shaking water bath at 37° C. Aliquots (100n1) were withdrawn in triplicate at 0, 15, 30, and 60 minutes andcombined with 900 μl of ice-cold 50/50 acetonitrile/dH₂O to terminatethe reaction. Each sample was analyzed via LC/MS/MS. The natural log ofthe percent remaining was plotted versus time. A linear fit was used todetermine the rate constant. The fit was truncated when percentremaining of test article was less than 10%. The elimination half-livesassociated with the disappearance of test and control articles weredetermined to compare their relative metabolic stability.

Genotoxicity

An exploratory bacterial mutagenicity assay (Ames test) was used toassess the potential of the compound genotoxicity. This assay utilizedS. typhimurium tester strains TA7007 and TA7006 (single base pairmutations) and TA98 (frame shift mutation) with and without metabolicactivation (Arochlor-induced rat liver S9) as described previously.³²

Pharmacokinetic (“PK”) Assessments in Rats and Newborn Mice

Analysis of the oral pharmacokinetics of selected compounds wasperformed in Sprague Dawley rats in a single dose study with serumsamples taken over a 24 h period. For the newborn mice PK evaluation, 4day old BALB/c mice were dosed intraperitoneally (IP) and serum sampleswere taken over a 24 hour period. A 50 μl aliquot of plasma was combinedwith 150 μl of 100% acetonitrile containing an internal standard (100ng/ml tolbutamide) in a 1.5 ml centrifuge tube. Samples were vortexedand centrifuged at 13,000 rpm for ten minutes. An 80 μl aliquot of theresulting supernatant was then transferred to an HPLC for vial analysis.Plasma levels of each compound were determined by LC/MS/MS, andpharmacokinetic parameters were determined using WinNolin software.

Efficacy in Newborn Mouse Model

To determine tolerability of ST-294, newborn (4 days old) BALB/c micewere given IP dosages of 0 (vehicle), 10, 25, or 100 mg/kg/day of ST-294for 5 days with assessment of clinical status daily.

To test the efficacy of ST-294 in the Tacaribe newborn mouse model, fourday old BALB/c mice (8 per dose group) were challenged with 3×10³ PFU(30XLD₅₀) of Tacaribe virus per mouse by IP injection with death as theend point. Mice were either treated with placebo (vehicle), ribavirin(MP Biomedical) administered IP at 25 mg/kg once a day for 10 days, orST-294 administered IP at 100 mg/kg once a day or at 50 mg/kg twice aday for 10 days. Mice were monitored daily and weighed every other daythroughout the study. Any mice showing signs of morbidity wereeuthanized by CO₂ asphyxiation. All animal studies conformed to theInstitute for Laboratory Animal Research and were approved throughappropriate IACUC review.

Results Homology Between Tacaribe and Other BSL 4 NWA

There are currently 23 recognized viral species of the Arenaviridaefamily.⁴ These viruses have been classified into two groups: the OldWorld (Lassa/LCM) arenaviruses and the New World (Tacaribe complex)group. The New World Tacaribe complex comprises three phylogeneticlineages, designated clades A, B, and C. Clade B includes the prototypicTacaribe virus, Amapari virus and the four South American Category Apathogens (Junin, Machupo, Guanarito and Sabiá). Tacaribe virus is 67%to 78% identical to Junin virus at the amino acid level for all fourviral proteins.²³ Working with authentic Category A arenavirusesrequires maximum laboratory containment (BSL-4), and therefore presentssignificant logistical and safety issues. Since Tacaribe virus isclosely related to the Category A pathogens it was chosen as a surrogateBSL 2 NWA for the development of a HTS assay to screen for inhibitors ofvirus replication.

Tacaribe HTS Assay

Since Tacaribe virus grows well in cell culture and causes clearvirus-induced cytopathic effect (CPE) a robust HTS CPE assay wasdeveloped in a 96-well plate. The CPE assay is a whole cell assay whichallows for calculation of the selective index of the compounds andidentification of inhibitors of any essential steps in the virus lifecycle. Of the 400,000 compounds screened in the Tacaribe virus HTSassay, 2,347 hits were identified (0.58% hit rate). All of these hitshad EC50 values ≦5 μM. The 2,347 hits were then qualified based on fourcriteria: i) chemical tractability, ii) inhibitory potency, iii)inhibitory selectivity, and iv) antiviral specificity. A chemicallytractable compound is defined as an entity that is syntheticallyaccessible using reasonable chemical methodology, and which possesseschemically stable functionalities and potential drug-like qualities.Hits that passed this medicinal chemistry filter were evaluated fortheir inhibitory potency. EC₅₀, CC₅₀, and selective index (SI) valueswere determined to assess whether the hit was a selective inhibitor.Hits with SI values of at least 10 were considered further. Of the 2,347hits identified, 36 compounds exhibited all the characteristics ofquality hits. These compounds were chemically tractable, had EC₅₀ values≦5 μM and SI values ≧10. Among the 36 quality hits, there were severalclusters of structure type. One structure type was chosen for furtherdevelopment and ST-336 is the representative prototype for this series.ST-336 is a 407.33 dalton compound and its structure is shown in FIG. 1.

TABLE 1 Specificity of ST-336 Virus (assay) ST-336 (μM) NWA Tacaribe(CPE) EC50    0.055 (CPE) EC90    0.125 (Virus yield) EC90    0.068(Virus yield) EC99    0.085 (Plaque reduction) EC50    0.100 Candid1(CPE) EC50    0.062 Amapari (CPE) EC50 <20* Machupo (Plaque reduction)EC50    0.150 Guanarito (Plaque reduction) EC50    0.300 Junín (Plaquereduction) EC50    0.150 OWA Lassa (Plaque reduction) EC50 >20 LCMV(Elisa) EC50 >20 Results represent the average of at least twoindependent determinations. *20 μm represents limit of compoundsolubility

Characterization of ST-336

As seen in Table 1, ST-336 has submicromolar potency, good selectivity,and antiviral specificity against Tacaribe virus as well as the CategoryA NWA. Evaluation of ST-336 in a virus yield reduction assay againstTacaribe virus produced EC %) and EC₉₉ values of 0.068 μM and 0.085 μMrespectively. The CC₅₀ value for ST-336 on Vero cells is >20 μM whichrepresents the solubility limit of this compound in cell culture media,giving it a selective index of >363. The activity of ST-336 againstTacaribe virus was tested on multiple cell lines and all the EC₅₀ valueswere similar to those achieved on Vero cells (data not shown). Whentested against several arenaviruses, ST-336 showed no inhibitoryactivity against OWA, either LCM virus or authentic Lassa virus (Table1). This drug also lacked activity against the NWA Amapari virus. Thiswas a surprising result given the close phylogenetic relationshipbetween Amapari and Tacaribe viruses.²³′ ¹⁹ This discrepancy is laterdiscussed following sequencing of GP2 of all NWA. However, importantlyST-336 showed potent antiviral activity against the vaccine strain ofJunin virus (Candid 1) as well as Machupo, Guanarito, and Junin (Table1).

TABLE 2 Selectivity of ST-336 Virus (assay) ST-336 EC50(μM) DNA virusesHSV-1 (CPE) >20* CMV (Elisa) >20 Vaccinia (CPE) >20 RNA viruses RSV-A(CPE) >20 Rotavirus (CPE) >20 SARS (CPE) >20 Ebola (CPE) >20 Resultsrepresent the average of at least two independent determinations. *20 μmrepresents limit of compound solubility

The specificity of the antiviral activity exhibited by ST-336 wasdetermined by testing against a number of related and unrelated viruses.As shown in Table 2 above, ST-336 showed no activity against a varietyof unrelated DNA (HSV, CMV, vaccinia virus) and RNA (RSV, Rotavirus,SARS and Ebola virus) viruses.

Mechanism of Action of ST-336

A single cycle (24 h) time of addition experiment was done to determinewhen during the virus replication cycle ST-336 exerts its antiviralactivity. The effect of ST-336 on Tacaribe virus yield was determinedfollowing addition of compound to Vero cell cultures at various timesbefore or after infection. ST-336 was added at one hour before infection(-1 h), during virus adsorption (Oh), and at several timespost-infection. Drug was kept, following sequential addition, oninfected cell cultures for the entire time of the experiment. Controlinfected cultures were treated with drug vehicle (DMSO) only. At 24hours post-infection, samples were collected, and virus yields weredetermined by plaque assay. As shown in FIG. 2A, ST-336 exerted itsinhibitory effect only at the very early stage in the virus life cycle.Addition of ST-336 at any time points post-infection had no effect onvirus yield. These data suggest that ST-336 is an early stage inhibitorof virus replication.

These results were confirmed in a second type of time additionexperiment. In this experiment, compound was spiked in the culturemedium for only 1 hour, at 1 hour before infection (−1 h), duringinfection (0) and at 1 hour post infection (+1 h), and then removed. Thecultures were washed to remove any residual compound and overlaid withagarose. Virus plaque numbers were then determined at 5 dayspost-infection. Data in FIG. 2B showed that while compound added beforeand after virus adsorption for 1 hour had no effect on plaque formation,compound added during the 1 h adsorption/entry process dramaticallyreduced Tacaribe plaque formation. These data are consistent with ST-336being an adsorption/entry inhibitor.

Two approaches were taken to determine if ST-336 is binding to intactvirions. In the first experiment, 1000 PFU of purified Tacaribe viruswas incubated with ST-336 or DMSO and dialyzed overnight at 4° C. andtitrated. While no virus was titrated from the dialyzed bag originallyincubated with drug, more than 300 PFU of virus was titrated from theDMSO vehicle dialyzed bag (data not shown). No drug was biologicallydetected in the dialysis bag originally containing 5 μM of drug asmeasured by the incapability of the virus plus drug dialyzed mixture toinhibit freshly added Tacaribe virus (300 PFU). These data suggestedthat ST-336 binds intact virions with a very slow dissociation constant.In the second experiment (FIG. 3), Tacaribe virus was incubated in atest tube with 5 μM of ST-336 or DMSO. Serial 1:10 dilutions wereperformed and for some samples ST-336 was added as a specified dilutionrepresenting the concentration of drug expected following sampledilution. As virus and compound are diluted with media, the compoundconcentration will reach a concentration without an inhibitory effect,unless the compound was capable of binding to virus. Test virus withoutcompound in the initial tube was also diluted in media and compoundconcentrations corresponding to that found in the tubes where virus andcompound were diluted together was added to each virus dilution.Titration on Vero cells showed that ST-336 present in excess in theinitial tube was carried over for two additional 1:10 dilutions throughspecific virus binding and inhibits virus infection. Whereas when drugwas added at a specified dilution virus was not inhibited to the samedegree as virus diluted with drug (data not shown). These data suggestthat ST-336 binds with at least a slow K_(off) to intact protein presenton Tacaribe virus.

Isolation of Drug Resistant Variants

The expected mutation rate of RNA viruses is very high (˜1 mutant in10,000) and a common approach to determining the target of an antiviralis to isolate virus resistance to the antiviral and then map the site ofresistance. Virus variants with reduced susceptibility to ST-336 wereisolated from wild type Tacaribe virus stocks plated in the presence ofST-336. The observed frequency of ST-336 drug resistant (ST-336^(DR))variants was as expected for RNA viruses. Sixteen ST-336^(DR) isolatesfrom four independent wild type Tacaribe virus stocks were isolated andplaque purified three times. All ST-336^(DR) isolates were tested fortheir ability to grow in the presence of ST-336. The growth ofST-336^(DR) isolates was unaffected by the presence of ST-336 atconcentrations that completely inhibited wild type Tacaribe virusreplication (data not shown). The isolation and confirmation of drugresistant virus variants strongly suggest that ST-336 acts as a directantiviral inhibitor.

To determine the genetic basis for resistance and the molecular targetof ST-336, RNA was isolated from the wild type and ST-336^(DR) isolates.Based on the time of addition experiments, it was suspected that theviral glycoproteins might be the target of ST-336. The entireglycoprotein precursor GPC region of the S segment was sequenced.Sequence analysis was performed on four wild type isolates (WT#1-4) andfour ST-336^(DR) isolates derived from drug selection applied to eachcorresponding parental wild type isolate (DR#1.1 from WT#1, DR#2.1 fromWT#2, DR#3.1 from WT#3 and DR#4.1 from WT#4). The sequence analysisshowed that the GPC gene from the four parental wild type isolates hadidentical sequences. When compared to the GPC sequences of four drugresistant variants, each possessed a single nucleotide change that inall cases resulted in an amino acid change. FIG. 4A shows the locationof each of the mutations which are located in or around thetransmembrane domain of GP2. The sequence alignments of the region ofthe GP2 containing the changes is presented in FIG. 4B. The singlechange in DR#1.1 was at amino acid position 418 (1418T), in DR#2.1 atamino acid 416 (T416N), in DR#3.1 at amino acid 433 (S433I) and inDR#4.1 at amino acid 436 (F436I). 1418 is similarly conserved (I or L,but never a T) in all Glade B New World arenavirus, while T416 isconserved among all Glade B NWA. F436 is similarly conserved with oneexception; Amapari virus encodes a leucine at position 436. This changein Amapari virus may explain its lack of susceptibility to ST-336 (Table2). 1418, T416, 5433 and F436 lie near the N-terminal and C-terminallimits of the putative transmembrane domain of GP2, a region known toplay a vital role in enveloped virus fusion.^(17,27,28,38,39) Takentogether, these data suggest that amino acid changes in arenavirus GP2at either position 416, 418, 433 or 436 are sufficient to confer reducedsusceptibility to ST-336 and are consistent with the proposed fusioninhibition mechanism suggested by virological experiments.

Hit-to-Lead Optimization

Preliminary data showed that ST-336, while demonstrating interestingantiviral activity and specificity, had poor pharmacokinetic (PK)properties in rodents (mouse and rats, data not shown). In order toimprove the PK properties of ST-336, a lead optimization chemistrycampaign was initiated. The objective of the optimization program was todevelop compounds that possess attributes consistent with the ultimatedrug product profile. Lead optimization activities comprised a series ofiterations involving design and chemical synthesis of analogs of thelead structure, followed by a series of biological, physiochemical, andpharmacological evaluations of the new compounds. Chemical analogsflowed through a compound evaluation paradigm that involved first invitro virological and cytotoxicity assessments, followed by a series ofevaluations as listed: in vitro metabolic stability (S9), solubility,exploratory bacterial mutagenesis and pharmacokinetic assessments. 165analogues were prepared and the most potent were examined for in vitrometabolism in S9 liver extracts. The most stable were dosed in rats, andST-294 emerged as a potent, orally bioavailable representative of thecompounds.

Characterization of ST-294

The structure of ST-294(N-2-(1,1,1,3,3,3-hexafluoro-1-methylpropyl)-2-[(4-difluoromethoxyphenyl)sulfonyl]hydrazine-1-carboxamide)is show in FIG. 5. ST-294 was tested against the drug resistant Tacaribemutants generated with ST-336 (DR#1-4) and all of the mutants elicitedcross-resistance to ST-294 suggesting that this compound is targetingthe same area of GP2 as ST-336 (data not shown). The activity of ST-294against Tacaribe, Machupo, Guanarito, and Junin viruses was similar tothat seen with ST-336 (Table 3). The CC₅₀ of ST-294 on Vero cells is >50μM yielding a selective index of >416. Further characterization ofST-294 showed that this compound is soluble up to 23 μM in mediacontaining 10% fetal calf serum and up to 480 μM in buffer at pH 7.4(Table 3). The metabolic stability of ST-294 was tested in S9 liverextracts from rat, mouse, human, and guinea pigs and was found to bemost stable in human S9 followed by mouse, rat and guinea pigrespectively (Table 3). Analysis of the oral pharmacokinetics of ST-294was initially performed in the rat as this species is well characterizedfor this type of study. The rats were dosed with ST-294 by oral gavageand samples were taken over a 24 h period. Serum levels were very high(C_(max)=6670 ng/ml) and ST-294 has good oral bioavailability (68.2%)(Table 3).

TABLE 3 Characterization of ST-294 Virus (assay) ST-294 Tacaribe (CPE)EC50 0.120 μM (Plaque reduction) EC50 0.100 μM Machupo (Plaquereduction) EC50 0.300 μM Guanarito (Plaque reduction) EC50 1.0 μM Junin(Plaque reduction) EC50 0.300 μm Properties Solubility (0%, 2%, 10% FBS)18, 21 and 23 uM Solubility (pIon, pH 7.4) 480 μM Stability (S9)rat/mouse/human/g.p 26/74/100/23 min Genotoxicity (Ames test) negativePK (rat/oral) ½ life 2 hours bioavailability (F) 68.2% PK (newbornmouse/IP) ½ life 3 hours C_(max) 2910 ng/mlEfficacy Study with ST-294 in Newborn Mouse Model

ST-294 has potent antiviral activity against NWA and good drug-likeproperties, so the next step was to test the ability of ST-294 toinhibit NWA-induced disease in an animal model. For the Category Aagents, the experiments require BSL 4 containment. However, in an effortto obtain an initial readout, a Tacaribe virus challenge model innewborn mice was established. In preparation for this study, PK andtolerability experiments were performed with ST-294 in newborn miceprior to conducting an efficacy trial. Newborn (4 day old) BALB/c micewere dosed IP with 10 mg/kg of ST-294 and blood samples were collectedfor analysis. Relative to in vitro antiviral concentrations required toinhibit Tacaribe virus CPE (EC₅₀=66 ng/ml), mean plasma concentrationsin newborn mice were well above this level for prolonged periods of time(>15× through 8 h and 6× at 24 h after dosing, data not shown). In thismodel the drug is delivered via the IP route due to the difficulty ofperforming multiple oral gavages on newborn mice. To test tolerability,newborn mice were given IP dosages ranging from 0-100 mg/kg/day ofST-294 for 5 days. Dosages of 100 mg/kg/day for 5 days were welltolerated by the newborn mice as there were no clinical signs oftoxicity and the mice gained weight at the same rate as the control mice(data not shown). This highest tested concentration of ST-294 of 100mg/kg/day was used in a Tacaribe animal efficacy study.

The drug levels and half-life shown in the PK study in the newborn micewas not equivalent to that seen in the rats, but the serum levels seemedsufficient to perform a proof-of-concept animal study in the Tacaribeanimal model. Four day old mice were challenged with 30×LD50 of Tacaribevirus and treated with placebo, ribavirin as a control or ST-294. As theresults in FIG. 6 demonstrate, ST-294 showed efficacy in the Tacaribeinfected newborn mice with both survival and a delay in death similar tothe drug control (ribavirin). Taken together these data suggest thatST-294 is a promising and appropriate drug candidate to advance intodefinitive animal studies where guinea pigs and primates will bechallenged with authentic NWA (Junin and Guanarito viruses) and treatedat various times post infection and prophylatically with ST-294.

DISCUSSION

Through a successful HTS and medicinal chemistry program, a NWAantiviral drug candidate, ST-294, has been identified. This drugpotently and selectively inhibits NWA viruses in vitro including the 3NIAID/CDC Category A viruses (Junin, Machupo, and Guanarito viruses).This compound was also evaluated for stability in S9 liver extracts andfor it's pharmacokinetic properties and was found to be metabolicallystable and orally bioavailable. In a preliminary animal efficacy study,ST-294 showed significant protection against Tacaribe virus induceddisease in newborn mice. Through mechanism of action studies it isapparent that this series of compounds targets GP2 and are viral entryinhibitors.

From the dialysis and dilution experiments (FIG. 3) it is apparent thatthe drug binds to virus and is carried over during dilutions. Thisphenomenon could potentially have an effect when titrating virus samplesduring other experiments. However, in the time of addition experiment,there was not enough drug carry over due to high dilution to affect thetiters when added 1 hour or more after infection (FIG. 2).

Since ST-294 has better S9 stability than ST-336 does, it is thoughtthat metabolism occurs at the methyl group on the aromatic ring (FIG.1). The benzylic position is susceptible to oxidation. When there is nobenzylic hydrogen present as in ST-294 (FIG. 2), the oxidation isblocked and thus eliminates the fastest metabolism pathway. The additionof the difluoromethoxy group in ST-294 gave this compound increased S9stability, but did not reduce antiviral activity.

In the Tacaribe newborn mouse model the mice appear to die of aneurological disease (indicated by hind quarter paralysis) and it is notknown whether ST-294 can cross the blood brain barrier. Also the druglevels and half-life of this drug candidate given IP in newborn mice isnot as good as oral dosing in rats so serum levels and compound gettingto the brain may have compromised the ability to obtain completeprotection in this model. The more appropriate animal models forhemorrhagic fever caused by arenaviruses are in guinea pigs andnon-human primates where the virus replicates predominantly in thespleen, lymph nodes and bone marrow causing hemorrhagic diathesis.Guinea pig models are well established for Junin, Machupo, and Guanaritovirus diseases, and represent the best small animal model for evaluationduring preclinical studies.^(26,34) Guinea pigs infected with pathogenicstrains of Junin virus develop a fatal disease akin to human AHF.³⁷

There are many reports of the role of transmembrane in the function ofviral fusion proteins. In the case of influenza virus hemagglutinin, itis clear that a transmemebrane anchor is required for full fusionactivity.²⁷ In contrast, specific sequence requirements within thetrasmembrane domain have been identified, for example, in humanimmunodeficiency virus (HIV) type 1, murine leukemia virus, foamyviruses, coronavirus, Newcastle disease virus and measles virus.²⁷ Basedon the drug resistant variants generated during these studies, theST-336 class of compounds targets the GP2 envelope protein, withmutations eliciting reduced susceptibility to the drug arising in oraround the transmembrane region (FIG. 4).

Drugs that target the interactions between the virus envelope and thecellular receptor represent a new class of antiviral drugs. For HIVtherapy, entry inhibitors have recently raised great interest because oftheir activity against multi-drug resistant viruses. A new antiviralagainst HIV was recently approved by the FDA called enfuvirtide.Enfuvirtide (Fuzeon) is a potent fusion inhibitor that blocks formationof the six-helix bundle and thus prevents membrane fusion.²⁹ Enfuvirtidehas been successful in improving the virological and immunologicalresponse in treatment-experienced HIV-infected patients.³³ There areseveral other compounds that counter HIV entry that are in differentdevelopmental stages, among them: 1) the attachment inhibitordextrin-2-sulfate; 2) the inhibitors of the glycoprotein (gp) 120/CD4interaction PRO 542, TNX 355 and BMS 488043; and 3) the co-receptorinhibitors subdivided in those targeting CCR5 or CXCR4.²⁰ The success ofenfuvirtide and others in the development pathway are proof that virusentry inhibitors can be used to treat viral diseases in humans.

ST-294 also has the potential for prophylactic use since this drugappears to bind to the virus (FIG. 3) and would prevent infection. Othervirus entry inhibitors have demonstrated protection when givenprophylactically.²² This is an indication that can be i pursued todetermine its feasibility.

The results presented here show that ST-294 is a potent specificinhibitor of New World arenaviruses including the Category A hemorrhagicfever viruses (Junin, Machupo, and Guanarito). More importantly, thetarget of ST-294 (virus entry into the cell) serves as a viable targetfor antiviral development. Since virus infection can be completelyinhibited at concentrations in the nanomolar range, the target forST-294 would seem to be both accessible and extremely sensitive toreagents that disrupt its role in the infection process. Therefore, itwill be important to further define the mechanism involved in ST-294mediated inhibition.

Pseudotyped Antivirus Assay.

Viral pseudotypes were used to antiviral activity of the compounds99-116. This is a surrogate assay that uses only the envelope protein ofthe target virus, not the virus itself. Viral pseudotypes are generatedby cotransfection of a replication-defective retroviral proviruscontaining a reporter gene, and a plasmid expressing a heterologousviral envelope. The provirus is engineered so that the homologousretroviral envelope is not expressed, and thus heterologous viralenvelope proteins are acquired as budding viral particlesnonspecifically capture cell surface proteins. Pseudotypes prepared inthis manner will infect cells via the heterologous envelope and arecommonly used to assay functions of the heterologous envelope.

Another use of viral pseudotypes is that they allow functional analysisof an envelope outside of the context of the virus from which it wasderived. Many hemorrhagic fever viruses require maximum laboratorycontainment (BSL-4), which impart significant logistical and safetyissues. Surrogate assays can be performed under less-restrictive BSL-2laboratory conditions, since they do not use the pathogen itself. Thisstrategy was used to examine antiviral efficacy against hemorrhagicfever arenaviruses that normally require maximum laboratory containment,such as Machupo and Guanarito viruses.

Pseudotype virus infection was assayed in tissue culture cells,specifically, 293T (human embryonic kidney). Cells are seeded into96-well plates so that they are somewhat subconfluent on the followingday. In order to test the inhibitory properties of a given compound,serial compound dilutions were prepared in DMSO. Each of these dilutionswas then further diluted 100-fold in cell culture media. Further, cellculture media was replaced with the compound dilutions in media, andthen subsequently an equal volume of pseudotype virus stock was added.

The pseudotype virus was diluted in cell culture media such that theamount of virus added to each well was sufficient to produce aluciferase signal providing a signal-to-noise ratio of 20 to 50.Luciferase activity was measured 2-3 days post-infection using standardluciferin-based bioluminescence detection methods, such as Promega'sLuciferase Assay System. Each compound dilution was tested in triplicatewells, and luciferase activity was converted to a percentage ofinfectivity based on positive (no compound) and negative (no virus)controls on the same plate. Fifty percent effective concentrations(EC₅₀s) were calculated with IDBS XLfit4.1 software for Microsoft Excel,using a four parameter logistic model fit to y=A+B/(1+(x/C)̂1)), where A(bottom) and B (top) are fixed at 0 and 100% respectively, C=EC₅₀,D=slope factor, x=compound concentration, and y=response. The resultsare summarized in Table C.

TABLE C Antiviral activity vs. pseudotyped viruses A: EC₅₀ ≦1 μM; B: <EC₅₀ < 10 μM VSV (μM) Compound Tacaribe Guanarito Machupo Junín(negative control) 99 A A A >30 100 A A A 19.7 101 A A A >30 102 A AA >20 103 A A A >20 104 A A A >30 105 A A A >30 106 A A A >30 107 A AA >30 108 A* 109 A B B >30 110 A B A A >30 111 A A A >30 112 A B A >30113 A A A 21 114 A A A A >30 115 A A A >30 116 A B A >20 A* for compound108 is the result of cytopathic effect assay

the Use of ST-375, a Dansyl Analogue of ST-294 to Detect Arenavirus.

Antiviral strategies to interfere with virus entry into the host cellhave in many instances proven successful in preventing virus infectionand mitigating disease. Arenavirus entry takes place in the endosomethrough a process of pH-dependent membrane fusion, mediated by the virusenvelope glycoprotein (GPC)⁴⁰. GPC is unique among class I viral fusionproteins 41, 42 in that the mature complex contains a stable signalpeptide (SSP) in addition to the prototypical receptor-binding andtransmembrane fusion subunits (G1 and G2, respectively)⁴³. Interactionsbetween the ectodomains of SSP and G2 are thought to play a role inmaintaining the prefusion form of GPC at neutral pH, and activating theconformational changes leading to membrane fusion at acidic pH⁴⁴

Small-molecule compounds that specifically inhibit GPC-mediated membranefusion have recently been identified using high-throughput screening(HTS) methods⁴⁵⁻⁴⁸. These lead compounds, as summarized by FIG. 7,comprise six chemically distinct classes and exhibit differentspecificities for New World (NW) and/or Old World (OW) arenavirusspecies. Among the compounds, ST-294 and ST-193 have been shown toprotect against lethal arenavirus infection in animal models⁴⁵ Thegenetic studies of GPC from the Junin virus (JUNV), the causative agentof Argentine hemorrhagic fever, suggest that these small-moleculeinhibitors bind to and stabilize the prefusion GPC complex, therebypreventing pH-induced activation of membrane fusion and virus entry.⁴⁸

Recombinant baculoviruses provide a robust platform for high-levelexpression of membrane glycoproteins⁴⁹ and thus the intact transmembraneGPC complex of the JUNV was expressed in insect cells. For theseexperiments, recombinant baculoviruses were generated using theInvitrogen Bac-to-Bac™ system. Coding regions corresponding to SSP andthe G1G2 precursor of GPC from the pathogenic MC2 strain of JUNV(GenBank accession number D10072)⁴³′⁵³ were inserted downstream of thebaculovirus p10 and polyhedrin protein promoters in the pFastBac-Dualvector (Invitrogen), respectively. The G1G2 precursor was expressedusing the conventional signal peptide from human CD4⁴³. In this mode ofGPC expression, SSP and the G1G2 precursor are translated independentlyand associate in trans to reconstitute the native GPC complex^(57,58)The SKI-1/S1P processing site in the G1G2 precursor was mutated toprevent proteolytic maturation⁴³ and a FLAG-tag sequence was appended tothe C terminus to facilitate purification. Previous studies had shownsimilar C-terminal tags to be innocuous^(43,54,55). Bacmids weregenerated using Escherichia coli DH10Bac cells (Invitrogen) and thesewere used to transfect Spodoptera frugiperda Sf9 cells (Invitrogen) togenerate the recombinant baculovirus.

Further, Baculoviruses encoding the recombinant cleavage defective GPC(icd-GPC) were used to infect Trichopulsia ni High-Five™ cells(Invitrogen) for expression and protein purification. Cultures wereinoculated with the P3 virus stock at a density of 2×10⁶ cells/ml andallowed to grow at 27° C. for 48-52 hours. The cells were pelleted,frozen in liquid nitrogen, thawed and resuspended in lysis buffer (25 mMTris, 250 mM NaCl, 2 mM MgCl₂, 100 Mμ ZnCl₂ and protease inhibitors, pH7.4). Nitrogen decompression (Parr Bomb) was used to disrupt cells,which were then subjected to a low-speed spin to remove cellular debris.The membrane fraction was recovered by ultracentrifugation at 100,000×gfor 1 hr. The pellet was resuspended in high-salt lysis buffercontaining 450 mM NaCl and again recovered by ultracentrifugation.Membranes were solubilized in lysis buffer containing 150 mM NaCl and1.5% dodecyl-β-D-maltoside (DDM) using a Dounce homogenizer. The lysatewas stirred for 2 hr, clarified (100,000×g for 1 hr), and thesupernatant was incubated with M2 anti-FLAG MAb immobilized to agarosebeads (Sigma) for 2 hr with slight agitation. The beads were then loadedonto a column, washed with DDM-containing lysis buffer to removenon-specifically bound proteins, and icd-GPC was eluted with 5 μM of3×FLAG peptide (Sigma). The eluate was dialyzed to remove the peptideand subjected to size-exclusion chromatography using a Superdex-200/G-75tandem column (GE Healthcare).

Gel filtration was also used to exchange detergents and vary DDMconcentrations. A panel of detergents of varying hydrophobic/hydrophilicproperties, lipid chain length and head groups were investigated tooptimize for retention of the trimeric state of icd-GPC. Detergents(Anatrace) included the following β-D-maltosides in addition to DDM:n-tridecyl-, n-tetradecyl-, n-octyl-, n-undecyl-,5-cyclohexyl-1-pentyl-(Cymal-5), and 6-cyclohexyl-1-hexyl-(Cymal-6).Others tested include: nonanoyl-N-methyl glucamide (Mega-9),decanoyl-N-methyl glucamide (Mega-10), nβoctyl-β-D-glucoside (OG),diheptanoyl phosphatidylcholine, and n-dodecylphoscholine.

Further, the studies determined whether isolated icd-GPC isantigencially similar to native GPC. Thus, in order to assess whethericd-GPC folds into a native conformation, the immunoprecipitationstudies were performed using a panel of five well-characterizedG1-directed MAbs raised against-inactivated JUNV virions. Four of theseMAbs (BE08, AG02, BF11, and AA09) have shown to be capable ofneutralizing viral infectivity and would serve as sensitive probes forthe native GPC conformation. As summarized by FIG. 8, all fiveG1-directed MAbs were able to immunoprecipitate icd-GPC, withefficiencies comparable to those seen using mammalian cleavage-defectiveGPC.

For these experiments, monoclonal antibodies specific to the GP1 (G1)subunit of the envelope glycoprotein (BE08, BF11, AA09, AG02, or BF09)or to the nucleoprotein (α-N) of Junin virus were used toimmunoprecipitate [³⁵S]-labeled lysates from either insect cells(Trichoplusia ni High Five™ cells from Invitrogen) infected withrecombinant baculovirus expressing recombinant C-terminally FLAG-taggedcleavage-defective Junin virus GPC (see top panel of FIG. 8) or fromVero cells expressing an identical construct except that it was untagged(see bottom panel of FIG. 8). Immunopreciptated proteins were separatedby SDS-PAGE and detected by autoradiography. Further, the top panel ofFIG. 8 indicates the presence of SSP (stable signal peptide), whichco-immunoprecipitates with icd-G1G2.

In both cases, MAb BE08 was less efficient than the others. GPC was notprecipitated by a control MAb (BG12) directed to the JUNV nucleoprotein(N). These studies indicate that icd-GPC is antigenicallyindistinguishable from the native GPC complex.

As icd-GPC retains many of the essential structural features of thenative GPC complex, it was important to determine whether therecombinant protein serves as a platform for physicochemical studies ofsmall-molecule fusion inhibitors (see FIG. 7). As previously mentionedand reviewed in FIG. 7, small molecule fusion inhibitor ST-294⁴⁵ andST-761 are specific to the NW arenaviruses while ST-193⁴⁶ and TSRI17C8⁴⁷inhibit both NW and OW viruses. These compounds, therefore, mightbe expected to bind JUNV GPC in vitro. By contrast, ST-161⁴⁸ and TSRI8C1⁴² are selective for the OW LASV.

For these experiments, binding to icd-GPC was initially examined indetergent-containing solution using a CM5 biosensor chip. As illustratedin representative sensorgrams and summarized by FIG. 9, all compoundscapable of inhibiting JUNV GPC-mediated membrane fusion bound toicd-GPC. For these experiments, baculovirus-expressed recombinantC-terminally FLAG-tagged cleavage-defective Junin virus GPC (icd-GPC)was purified from DDM (1.5% dodecyl-3-D-maltoside) lysates of infectedinsect cells (Trichoplusia ni High Five™ cells from Invitrogen) using ananti-FLAG mAb, captured by an anti-FLAG mAb immobilized (amine coupled)on a Biacore CMS chip in 0.1% DDM, and assessed for binding toinhibitors injected at either 100 μM (17C8 and 8C1) or 150 μM (allothers) as indicated by the labeled sensorgrams.

As shown in FIG. 9, no binding was observed using surfaces devoid oficd-GPC, or those containing the unrelated membrane protein aquaporin.Further, binding was also abolished in the presence of non-maltosidedetergents that disrupt the trimeric structure of the complex (notshown). Importantly, the LASV-specific inhibitors ST-161 and 8C1 did notbind to JUNV icd-GPC. Together, these studies validate the specificityand sensitivity of the biosensor measurements of inhibitor binding. Inconclusion, species selectivity among these diverse inhibitors correlatewith GPC binding, rather than with post-binding effects.

As shown in FIG. 7, ST-375, a1-[1,1-bis(trifluoromethyl)propyl]-3-[[5-(dimethylamino)-1-naphthyl]sulfonylamino]urea,is a dansyl analogue of ST-294 that inhibits JUNV GPC-mediated cell-cellfusion at an EC₅₀ similar to that of its parent (0.6 μM and 1.5 μM forST-375 and ST-294, respectively; see FIG. 10). As the fluorescentproperties of dansyl compounds are often sensitive to chemicalenvironment, and specifically to protein binding⁵² the furtherexperiments tested whether ST-375 binding to icd-GPC could be detectedas a change in dansyl fluorescence.

As showin in FIG. 10, ST-375 fluorescence at 525 nm did indeed increasein a time-dependent manner on binding to solubilized icd-GPC. For theseexperiments, Vero cells expressing recombinant Junin virus GPC andbacteriophage T7 RNA polymerase (effector cells) were co-cultured withtarget Vero cells infected with a vaccinia virus recombinant expressingβ-galactosidase under control of a T7 promoter. Cell-cell fusion wastriggered by adjusting cell culture medium to pH 5.0 and quantitated bymeasuring β-galactosidase activity 5 hours after returning culturemedium to neutral pH. Serial dilutions of test compound were added tothe co-culture 3 hours prior to induction of cell-cell fusion.Inhibition is plotted as a percentage of fusion in the absence ofinhibitor. Each line denotes fusion inhibition by a small molecule asindicated by the symbols: ST-294 (filled squares), ST-761 (opensquares), ST-193 (open circles), ST-375 (closed circles), and 17C8 (opendiamonds).

Further, the increase was delayed when ST-375 was added to preformedicd-GPC:ST-294 complex (see FIG. 10), and was unaffected by the additionof the unrelated molecule ST-545 (not shown). By contrast, as shown inFIG. 10, no change in fluorescence was observed in the absence oficd-GPC or on incubation of ST-375 with the unrelated membrane proteinaquaporin. Together, these data validate the use of dansyl fluorescenceto assess ST-375 binding to icd-GPC in solution.

Further experiments determined whether any of the chemically distinctclasses of fusion inhibitors shared a binding site with ST-375, as hadbeen suggested based on genetic studies of resistance to the SIGAcompounds^(46,48). Thus, consistent with this hypothesis, it had beendetermined that bound ST-375 was displaced from icd-GPC in a time- andconcentration-dependent manner by ST-294 as well as by ST-761 andST-193, but not by ST-161.

These results are summarized by FIG. 11. For these experiments,solubilized icd-GPC (1 μM) was incubated with 10 μM ST-375 in 0.1% DDMbuffer for 2 hours at 4° C.; unbound compound was removed byultrafiltration (Amicon) and the complex was readjusted to 1 μM.Further, non-fluorescent inhibitor compound was added to 10 μM asindicated by the labeled lines and the loss in dansyl fluorescence wasmeasured as a function of time in a PerkinElmer L55 luminometer (340 nmexcitation and 525 nm emission) equipped with a circulating water bathmaintained at 20° C. The time-dependent increase in dansyl fluorescenceupon initial ST-375 binding is shown in the insert; binding is delayedif ST-294 is pre-bound to icd-GPC, and no binding occurs if an unrelatedprotein (aquaporin) is substituted for icd-GPC.

Thus, despite its independent provenance, the broadly active TSRIinhibitor 17C8 was also able to displace ST-375 from icd-GPC, whereasthe LASV-specific compound 8C1 did not (see FIG. 11). Together, theseresults corroborate the specificity of the competitive binding assay,and the notion that the chemically distinct JUNV inhibitors share acommon binding site.

It is likely that the LASV-specific compounds ST-161 and 8C1 bind thehomologous site on LASV GPC. It had been previously showed that anST-294 resistant mutant of JUNV GPC (K33H) exhibits de novo sensitivityto ST-161⁴⁸. As with ST-161, the K33H mutation in SSP also renders JUNVGPC hypersensitive to 8C1 (see FIG. 10). Thus, all independently derivedSIGA and TSRI arenavirus entry inhibitors appear to be directed to acommon site on GPC.

These studies reveal a unique and highly vulnerable target forsmall-molecule inhibitors of GPC-mediated membrane fusion. Moreover,recombinant icd-GPC faithfully recapitulates the binding selectivitythat underlies species specificity among these chemically diversecompounds. These studies also demonstrate the use of dansyl fluorescenceto assess ST-375 binding to icd-GPC in solution, and thus demonstratethe feasibility of using dansyl fluorescence to detect the presence ofarenavirus envelope glycoprotein.

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All references cited herein are herein incorporated by reference intheir entirety for all purposes.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe invention.

1.-6. (canceled)
 7. A compound selected from the group consisting of:1-[1,1-bis(trifluoromethyl)propyl]-3-[(2,4,6-trimethylphenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-tert-butylphenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(2,5-dimethoxyphenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-(2-naphthylsulfonylamino)urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-isopropylphenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(5-chloro-2-methoxy-phenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(2-phenylphenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(3,4-dichlorophenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-bromophenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[[4-(difluoromethoxy)phenyl]sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(3,5-dimethylphenyl)sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[[5-(dimethylamino)-1-naphthyl]sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-(1-naphthylsulfonylamino)urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-methyl-2,3-dihydro-1,4-benzoxazin-7-yl)sulfonylamino]urea;1-[[2,5-bis(2,2,2-trifluoroethoxy)phenyl]sulfonylamino]-3-[1,1-bis(trifluoromethyl)propyl]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[[4-(thiadiazol-4-yl)phenyl]sulfonylamino]urea;1-[1,1-bis(trifluoromethyl)propyl]-3-[(5-bromo-2-methoxy-phenyl)sulfonylamino]urea;and1-[1,1-bis(trifluoromethyl)propyl]-3-[(4-phenylphenyl)sulfonylamino]urea.8. The compound of claim 7, wherein the compound is1-[1,1-bis(trifluoromethyl)propyl]-3[[4-(thiadiazol-4-yl)phenyl]sulfonylamino]urea.9.-20. (canceled)
 21. The compound of claim 7, wherein the compound is1-[1,1-bis(trifluoromethyl)propyl]-3-[[5-(dimethylamino)-1-naphthyl]sulfonylamino]urea.